SANTA CRUZ BIOTECHNOLOGY, INC.
Protocols
The Power to Question
Santa Cruz Biotechnology, Inc. provides an extensive range of high quality support products to complement our
broad range of primary antibodies. These include internally-produced secondary antibodies for Western blotting and
immunohistochemical applications, Luminol reagent, agarose-conjugated Protein A, Protein G PLUS and Protein L
for immunoprecipitation studies, nuclear extracts, whole cell lysates, tissue extracts, ready made blots for Western
blotting, Western blotting membranes, TransCruz™ gel shift oligonucleotides, blocking reagents and general lab supplies. Cruz Marker™ Molecular Weight Standards and Prestained Molecular Weight Standards are provided for use
in Western blotting procedures. Our ImmunoCruz™ Staining System is a pre-diluted, ready-to-use system for immunohistochemical staining of paraffin-embedded tissue sections. We have also introduced a series of Apoptosis Detection
Kits for the assessment of cells undergoing apoptosis. The reagents and recommended procedures for use of our
products are provided below and include (1) Western (immuno-) blotting, (2) immunoprecipitation, (3) immunoprecipitation/Western blots, (4) ExactaCruz™ immunoprecipitation/Western blots, (5) immune complex protein kinase
assays, (6) immunoperoxidase cell staining, (7) immunofluorescence cell staining, (8) flow cytometry (FCM), (9) ELISA
assays, (10) TransCruz™ gel supershift assays, (11) methods for the use of peptides to neutralize antibody activity,
(12) ChIP assays, (13) siRNA transfection, (14) semi-quantitative RT-PCR and (15) preparation of solutions.
1. WESTERN (IMMUNO-)BLOTTING
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A. Sample Preparation
NOTE: For a listing of available cell culture products including classical and specialty media, sera and media
additives, induction agents, antibiotics and attachment
agents, please see our catalog or visit our website at
www.scbt.com.
NOTE: For phosphorylation studies, lysates can be
enriched for phosphoproteins using Santa Cruz Biotechnology Inc.’s PhosphoCruz™ Protein Purification
System (sc-24964).
MONOLAYER CELLS
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Grow cells to subconfluency in a 100 mm x 20 mm
petri dish, remove culture medium and rinse cell monolayer with room temperature 1x PBS (10X liquid PBS:
sc-24946). The following steps should be performed
on ice or at 4° C using fresh, ice cold buffers.
Add 0.6 ml of RIPA buffer (sc-24948) to the monolayer
cells in the plate. Gently rock plate for 15 minutes at
4° C. Remove adherent cells with a cell scraper. Transfer the resulting lysate to a microcentrifuge tube.
SUSPENSION CELLS
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Collect approximately 2.0 x 10 cells by low-speed
centrifugation (e.g. 200xg) at room temperature for
5 minutes. Carefully remove culture medium.
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Wash the pellet with PBS at room temperature and again
collect by low-speed centrifugation. Carefully remove
supernatant.
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Add 1.0 ml of ice cold RIPA buffer (sc-24948) with freshly
added protease inhibitors and/or phosphatase inhibitors.
Gently resuspend cells in RIPA buffer with a pipet and
incubate on ice for 30 minutes.
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Further disrupt and homogenize cells by hydrodynamic
Wash the plate once with 0.3 ml of RIPA buffer and
combine with first lysate.
Optional: Add 10 µl of 10 mg/ml PMSF (sc-3597) stock
and/or pass through a 21-gauge needle to shear the
DNA.) Incubate 30–60 minutes on ice.
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Centrifuge cell lysate at 10,000xg for 10 minutes at
4° C. The supernatant fluid is the total cell lysate.
Transfer the supernatant to a new microcentrifuge tube.
This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA,
centrifuge and combine supernantants.
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shearing (21-gauge needle), dounce homogenization
or sonication, taking care not to raise the temperature
of the lysate. (Optional: Add 10 µl of 10 mg/ml PMSF
stock. Incubate 30 minutes on ice.
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Transfer to microcentrifuge tube(s) and centrifuge at
10,000xg for 10 minutes at 4° C. The supernatant fluid
is the total cell lysate. Transfer the supernatant to a
new microcentrifuge tube. This is your whole cell
lysate. For increased protein recovery, resuspend the
pellet in a small volume of RIPA, centrifuge and combine supernantants.
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Load up to 10 µl of lysate per 1.0 mm of well width for
gels of 0.75 mm thickness.
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We recommend the use of Cruz Marker™ molecular
weight standards (sc-2035). Load 2 µl/well for 0.75 mm
gels and 5 µl/well for 1.5 mm gels. When used with
Cruz Marker™-compatible secondary antibodies, internal standard bands will appear when the probed blot
is exposed to detection reagent. Alternatively, use
Prestained Molecular Weight Standards (sc-2361).
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NOTE: For phosphorylation studies, lysates can be
enriched for phosphoproteins using Santa Cruz Biotechnology Inc.’s PhosphoCruz™ Protein Purification
System (sc-24964).
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Weigh tissue and dice into very small pieces using a
clean razor blade. Frozen tissue should be sliced very
thinly and thawed in RIPA buffer (sc-24948) containing
protease inhibitors and/or phosphatase inhibitors. Use
3 ml of ice cold RIPA buffer per gram of tissue.
Further disrupt and homogenize tissue with a dounce
homogenizer or a sonicator, maintaining temperature at
4° C throughout all procedures. (Optional: Add 30 µl of
10 mg/ml PMSF (sc-3597) stock per gram of tissue.)
Incubate on ice for 30 minutes.
Transfer to microcentrifuge tubes, centrifuge at
10,000xg for 10 minutes at 4° C. Remove supernatant
and centrifuge it again. The supernatant fluid is the
total cell lysate. A longer centrifugation may be necessary to obtain a clear lysate.
C. Immunoblotting
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Block non-specific binding by incubating membrane
in Blotto (either Blotto A or Blotto B; IgG-free BSA,
sc-2323, is recommended when using anti-bovine
secondary antibodies) for 30–60 minutes at room
temperature. Alternatively, the membrane may be
blocked at 4° C overnight in a covered container,
using Blotto without Tween-20.
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If using a phospho-specific antibody, add 0.01% (v/v) of
each Phosphatase Inhibitor Cocktails A and B (sc-45044
and sc-45045) to the blocking solution and the antibody
diluent to inhibit phosphatases.
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Incubate the blocked membrane in primary antibody
diluted in Blotto for 1 hour at room temperature. (For
phospho-specific antibodies: Use Blotto B with 0.01%
(v/v) of each Phosphatase Inhibitor Cocktails A and B
(sc-45044 and sc-45045.) Optimal antibody concentration should be determined by titration. We recommend
a starting dilution of 0.5–2.0 µg/ml. Wash membrane
three times for 5 minutes each with TBST.
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Incubate the membrane for 45 minutes at room temperature with horseradish peroxidase (HRP) conjugated
secondary antibody or alkaline phosphatase (AP) conjugated secondary antibody, diluted to 1:500–1:2000
in Blotto. If high backgrounds are observed, secondary
antibody should be diluted further (up to 1:20,000). If
NOTE: For phosphorylation studies, lysates can be
enriched for phosphoproteins using Santa Cruz Biotechnology Inc.’s PhosphoCruz™ Protein Purification
System (sc-24964).
B. Electrophoresis
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Mix sample (40-60 µg whole cell lysate, 10-20 µg
nuclear extract, 10-20 µl transfected lysate or 10-20
ng purified protein per lane) with an equal volume of
2x electrophoresis sample buffer (sc-24945) and boil
for 2–3 minutes. Unused samples may be stored at
-20° C.
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Transfer proteins from the gel to a nitrocellulose or
PVDF membrane using an electroblotting apparatus
according to the manufacturer’s protocols.
NOTE: Ready-made blots of human or mouse whole
cell extracts or nuclear extracts or mouse tissues are
available as Cruz Blot Systems™.
TISSUE SAMPLES
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Electrophorese according to standard protocols.
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Cruz Marker™ molecular weight standards (sc-2035)
are used in the gel, the Cruz Marker™ compatible secondary antibodies must be used in order to visualize
standards with ECL.
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Pellet cellular debris by centrifugation at 10,000xg for
10 minutes at 4° C. Transfer supernatant to a fresh
microcentrifuge tube on ice.
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Preclear lysate by adding 1.0 µg of the appropriate
control IgG (corresponding to the host species of the
primary antibody), together with 20 µl of appropriate
suspended (25% v/v) agarose conjugate (Protein AAgarose, Protein G-Agarose, Protein A/G-Agaroseor
Protein L-Agarose). Incubate at 4° C for 30 minutes.
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Centrifuge at 3,000 rpm (e.g. Eppendorf 5415D; approximately 1,000xg) for 30 seconds at 4° C.
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Transfer the supernatantor approximately 100–1000 µg
total cellular protein, to a microcentrifuge tube. Add
1–10 µl (i.e. 0.2–2 µg) primary antibody (optimal antibody concentration should be determined by titration)
and incubate for 1–2 hours at 4° C. Alternatively, if
antibody agarose conjugate is employed, add 20 µl
(i.e. 5 µl packed beads) and incubate at 4° C for 1 hour
to over-night with mixing; skip the next step.
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Example: Following starvation, remove culture medium
and replace with methionine-free medium containing 5%
dialyzed fetal calf serum and 100 µCi/ml 35S-methionine.
Incubate 1 hour at 37° C. For some proteins a longer
labeling period (up to 18 hours) is preferable. Wash
cells with PBS as necessary to remove unincorporated
35S-methionine.
Add 20 µl of the appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein
A/G-Agarose or Protein L-Agarose). Cap tubes and incubate at 4° C on a rocker platform or rotating device
for 1 hour to overnight.
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Collect immunoprecipitates by centrifugation at
3,000 rpm (approximately 1,000xg) for 30 seconds at
4° C. Carefully aspirate and discard supernatant.
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NOTE: For a listing of available cell culture products including classical and specialty media, sera and media
additives, induction agents, antibiotics and attachment
agents, please see our catalog or visit our website at
www.scbt.com.
Gently wash pellet 2–4 times with 1.0 ml RIPA buffer
(more stringent) or PBS (less stringent), each time repeating centrifugation step above.
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After final wash, carefully aspirate and discard supernatant and resuspend pellet in 40 µl of 2x electrophoresis sample buffer (sc-24945).
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Boil samples for 2-3 minutes and subject to electrophoresis and autoradiography. Unused samples may
be stored at -20° C.
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Wash membrane three times for 5 minutes each with
TBST and once for 5 minutes with TBS.
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Incubate membrane in Chemiluminescence Luminol
Reagent (sc-2048) according to Luminol data sheetor
visualize proteins using standard protocols. If luminol
is used for visualization, an HRP-conjugated secondary
antibody must be used.
2. IMMUNOPRECIPITATION
NOTE: This procedure may be used for cells labeled with
radioactive compounds such as amino acids or orthophosphate. (Radioisotope use and disposal should conform to
institutional and governmental regulations.) Cell labeling
should be carried out in medium lacking the relevant
nonradioactive compound. Starving cells appropriately
prior to labeling is recommended. Incubate cultured cells
(80-90% confluent monolayer in 100 mm cell culture plate
or approximately 2–5 x 107 suspension cells in flask).
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Add 1–3 ml ice cold RIPA buffer (sc-24948) to subconfluent cell monolayer and incubate at 4° C for 10
minutes. For suspension cells, add the RIPA buffer to
washed cell pellet in a microcentrifuge tube.
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Disrupt cells by repeated passage through a 21-gauge
needle or sonication. Transfer to a microcentrifuge
tube.
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Optional: Wash cell culture plate with additional 1.0 ml
ice cold RIPA buffer and combine with original extract.
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Optional: After boiling, samples may be centrifuged to
pellet the agarose beads followed by SDS-PAGE analysis of the supernatant.
3. IMMUNOPRECIPITATION/WESTERN BLOTS
NOTE: For a listing of available cell culture products including classical and specialty media, sera and media
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additives, induction agents, antibiotics and attachment
agents, please see our catalog or visit our website at
www.scbt.com.
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Prepare a total cell lysate as described under Western
blot procedure.
NOTE: For phosphorylation studies, lysates can be
enriched for phosphoproteins using Santa Cruz Biotechnology Inc.’s PhosphoCruz™ Protein Purification
System (sc-24964).
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Preclear whole cell lysate (optional step) as follows.
To approximately 1 ml of whole cell lysate or tissue
extract, add 0.25 µg of the appropriate control IgG
(corresponding to the host species of the primary antibody), together with 20 µl of appropriate suspended
(25% v/v) agarose conjugate (Protein A-Agarose,
Protein G-Agarose, Protein A/G-Agaroseor Protein LAgarose). Incubate at 4° C for 30 minutes.
Pellet beads by centrifugation at 3,000 rpm (approximately 1,000xg) for 30 seconds at 4° C. Transfer
supernatant (cell lysate) to a new microcentrifuge
tube at 4° C.
To 1 ml of the above cell lysateor approximately
100–1000 µg of total cellular protein, add 10 µg of
primary antibody agarose conjugate (i.e. 5 µl volume
of packed beads) and incubate at 4° C for 1 hour to
overnight with mixing.
Alternatively, if primary antibody agarose conjugate is
not available, incubate 1 ml cell lysate with 1–10 µl
(i.e. 0.2–2 µg) primary antibody (optimal antibody
concentration should be determined by titration) for
1–2 hours at 4° C. Add 20 µl of appropriate agarose
conjugate suspension (Protein A-Agarose, Protein GAgarose, Protein A/G-Agarose or Protein L-Agarose).
Cap tubes and incubate at 4° C on a rocker platform
or rotating device for 1 hour to overnight.
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After final wash, aspirate and discard supernatant and
resuspend pellet in 40 µl of 2x electrophoresis sample
buffer (sc-24945).
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Boil samples for 2–3 minutes. Load up to 5–10 µl of
sample per 1.0 mm well width for gels of 0.75 mm
thickness.
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Continue with electrophoresis and immunoblotting as
described under Western blotting procedure.
NOTE: Depending on the secondary antibody that is
used, 55 kDa and 27 kDa heavy and light IgG chains,
respectively, of the primary antibody may be detected.
These bands will be less pronounced if a primary antibody agarose conjugate is used in the above procedure
or if ExactaCruz™ Reagents are used.
4. EXACTACRUZ™ IMMUNOPRECIPITATION/
WESTERN BLOTS
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Prepare a total cell lysate as described under Western
(Immuno-) blotting procedure.
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Preclear whole cell lysate (optional): To approximately
1 ml of whole cell lysate or tissue extract in a 1.5 ml
microcentrifuge tube, add 40-50 µl of the suspended
(25% v/v) IP matrix supplied with each kit. Incubate
for 30 minutes at 4° C while rotating.
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Pellet IP matrix via microcentrifugation at maximum
speed for 30 seconds at 4° C. Without disturbing pellet,
transfer desired supernatant (precleared cell lysate) to
a new microcentrifuge tube. Store precleared lysate
on ice and discard the pellet.
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Formation of the IP antibody-IP matrix complex: To a
microcentrifuge tube, add 40-50 µl of suspended (25%
v/v) IP matrix, 1–5 µg of IP antibody and 500 µl of PBS.
Optimal antibody amount should be determined by
titration. Incubate at 4° C on a rotator for at least 1
hour. This step can be performed in parallel with the
above preclearing step or performed the day before
and allowed to incubate overnight at 4° C.
Collect pellet by centrifugation at 3,000 rpm (approximately 1,000xg) for 30 seconds at 4° C. Carefully
aspirate and discard supernatant.
Wash pellet 2–4 times with either RIPA buffer
(sc-24948) (more stringent) or PBS (less stringent),
each time repeating centrifugation step above.
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NOTE: It is necessary that the species of the IP antibody matches the species of the IP matrix included
with each ExactaCruz™ kit. For example, when performing an IP with a mouse antibody, it must be incubated with the Mouse IP Matrix provided (sc-45040
or sc-45042).
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After incubation of the IP antibody with the species
specific IP matrix, pellet matrix via microcentrifugation
at maximum speed for 30 seconds at 4° C. Carefully
aspirate and discard supernatant.
Wash pelleted matrix 2 times with 500 µl of PBS, each
time repeating the above centrifugation and aspiration
steps.
Immunoprecipitation: After the final wash of the IP antibody-IP matrix complex, transfer lysate (100-1000 µg
of total cellular protein) to the pelleted matrix and incubate at 4° C on a rotator for 1 hour to overnight.
After incubation of the matrix and lysate, microcentrifuge at maximum speed for 30 seconds at 4° C to
pellet. Aspirate and discard supernatant or alternatively keep supernatant for another IP or testing via
western blot.
Wash pelleted matrix 2–4 times with either RIPA Lysis
Buffer: sc-24948 (more stringent) or 1x PBS (less stringent), each time repeating the above centrifugation
and aspiration steps.
After final wash, aspirate and discard the supernatant
and resuspend pellet in 40-50 µl of reducing 2x Electrophoresis Sample Buffer: sc-24945. Boil samples for
2–3 minutes. Note: The immunoprecipitated sample
must be completely reduced and denatured for ExactaCruz™ to work properly.
Perform a quick spin to pellet IP matrix and carefully
load supernatant onto gel. Continue with electrophoresis as described under the Western (Immuno) Blotting
procedure.
At this stage it is essential that the immunoblotting
(primary) antibody matches the species specificity of
the HRP conjugated ExactaCruz™ detection reagent
which is unique for each kit. Detect the immunoblotting (primary) antibody using the corresponding HRP
conjugated ExactaCruz™ reagent and Western Blot
Luminol Reagent: sc-2048.
NOTE: When using sc-45042 ExactaCruz™ E, the alternate immunoblotting protocol that is specific for this
kit must be followed as described below in order to
generate desired results.
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ExactaCruz™ E Alternate Protocol
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After transfer, block/wash membrane with TBST (10x
TBST: sc-24953) for 1 hour, changing TBST once half
way through the incubation.
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Dilute WB antibody with ExactaCruz™ E Dilution
Buffer (provided), add to membrane and incubate for
1–2 hours at room temperature.
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After incubation, wash 3x with 1x TBST, 5 minutes per
wash.
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Dilute ExactaCruz E Western Blot Reagent (1:10001:10000) with ExactaCruz E Dilution Buffer (provided),
add to membrane and incubate 1–2 hours at room
temperature.
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Wash membrane 3x with TBST, 5 minutes per wash.
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Wash membrane once with 1x TBS (10x TBS: sc-24951)
for 5 minutes.
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Incubate membrane in Western Blot Luminol Reagent:
sc-2048 according to Luminol data sheet.
5. IMMUNE COMPLEX PROTEIN KINASE
ASSAYS
NOTE: For a listing of available cell culture products including classical and specialty media, sera and media
additives, induction agents, antibiotics and attachment
agents, please see our catalog or visit our website at
www.scbt.com.
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Remove medium from 100 mm cell culture plate
(80–90% confluent monolayer) and wash once with
PBS.
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Add 1–3 ml ice cold RIPA buffer (sc-24948) to cell
monolayer and incubate at 4° C for 10 minutes.
(Note: the use of RIPA buffer may not be optimal for
some kinases. Composition of lysis buffer may need
to be optimized to maintain active kinase.)
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Disrupt cells by repeated passage through a 21-gauge
needle and transfer to microcentrifuge or 15 ml conical
centrifuge tube.
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Wash cell culture plate with addition of 1.0 ml ice cold
RIPA buffer, 0.5% Triton X-100 (Triton X-100: sc-29112)
and combine with original extract.
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Pellet cellular debris at 10,000xg for 10 minutes at 4° C.
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may also choose to analyze immobilized peptides prepared by standard methods or offered commercially.
Transfer supernatant to a new microcentrifuge or 15 ml
conical centrifuge tube at 4° C.
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Transfer 1.0 ml cell extract (supernatant from above
step) to a 1.5 ml microcentrifuge tube. Add 1-10 µl
(i.e. 0.2–2 µg) primary antibody (optimal antibody
concentration should be determined by titration) and
incubate for 1 hour at 4° C.
6. IMMUNOPEROXIDASE CELL STAINING
Add 20 µl of appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein
A/G-Agaroseor Protein L-Agarose). Cap tubes and incubate at 4° C on a rocker platform or rotating device
for 1 hour to overnight.
A. Tissue Culture Cells
Collect immunoprecipitates by centrifugation at 2,500
rpm (approximately 1,000xg) for 5 minutes at 4° C.
Carefully aspirate and discard supernatant.
NOTE: For a listing of available cell culture products including classical and specialty media, sera and media
additives, induction agents, antibiotics and attachment
agents, please see our catalog or visit our website at
www.scbt.com.
Suspend pellet in 20 µl of the appropriate protein
kinase assay buffer (e.g. 50 mM HEPES (HEPES:
sc-29097), 0.1 mM EDTA (EDTA: sc-29092), 0.01%
BRIJ® 35, 0.1 mg/ml BSA, 0.1% β-mercaptoethanol,
0.15 M NaCl. Buffer composition will depend upon the
kinase under study.
Add 10–1000 ng peptide substrate. Peptide substrate
concentration should be determined empirically for
the substrate/enzyme/cell line used.
Prepare 1 ml ATP mix: 930 µl appropriate protein kinase
assay buffer, 6 µl 50 mM ATP, pH 7.0, 20 µl 2.0 M
MgCl2 and 44 µl [γ P]-ATP [10 mCi/ml]. Add 10 µl ATP
mix per sample and incubate for 20 minutes at 30° C.
Place on ice.
Terminate the reaction by adding an equal volume of
2x electrophoresis sample buffer (sc-24945) and boil
samples for 2–3 minutes. After boiling, samples may
be centrifuged to pellet the agarose beads (optional);
the supernatant is analyzed. Analyze samples by SDSPAGE and autoradiography. Unused samples may be
stored at -20° C. Alternatively, labeled peptides can
be separated from unicorporated label by acid precipitation followed by collection on a filter and radioactivity determined by scintillation counting. Researchers
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Grow cultured cells on sterile glass cover slips or
slides overnight at 37° C. Wash briefly with PBS and
fix cells by one of the following procedures:
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Wash pellet four times with 1.0 ml RIPA buffer (more
stringent) or PBS (less stringent), each time repeating
centrifugation step above.
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NOTE: For a listing of cover glasses and micro slides for
Immunohistochemistry, please see our catalog or visit our
website at www.scbt.com.
1) 5 minutes in -10° C methanol, air dry (recommended
method); or
2) 2 minutes in cold acetone, air dry; or
3) 10 minutes in 1% formalin in PBS (keep wet).
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Wash in three changes of PBS.
Optional: Incubate for 5–10 minutes in 0.1-1% hydrogen
peroxide in PBS to quench endogenous peroxidase activity. Wash in PBS twice for 5 minutes each.
B. Frozen Tissue Sections
NOTE: For a listing of mounting media for Immunohistochemistry including Clarion and Crystal Mounting Media,
see our catalog or visit our website at www.scbt.com.
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Freeze tissue block in liquid nitrogen according to
standard procedures. Block may be stored at -70° C
for up to 2 weeks before sectioning.
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Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Or use pre-treated slides.
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Cut 4- to 10-micron thick sections. Adhere sections
to room temperature slides. Slides may be stored at
-70° C. Thaw slides at room temperature prior to fixing
and staining.
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Fix slides in cold acetone for 10 minutes and keep refrigerated (or choose other fixation procedure). Wash
in three changes of PBS.
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Optional: Incubate for 5–10 minutes in 0.1–1% hydrogen peroxide in PBS to quench endogenous peroxidase
activity. Wash in PBS twice for 5 minutes each.
saponin in deionized H2O at room temperature.
Wash at least three times in PBS. Aspirate excess
liquid from slides.
NOTE: For tissues containing high levels of endogenous
biotin (which may result in higher background staining),
we recommend following the Formalin-Fixed, ParaffinEmbedded Tissue Sections protocol, as endogenous
biotin is normally destroyed in paraffin-embedded
tissue.
Optional: Incubate for 5-10 minutes in 0.1-1% hydrogen peroxide in deionized H2O to quench endogenous
peroxidase activity. Wash in PBS twice for 5 minutes
each.
D. Immunoperoxidase Staining
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For immunoperoxidase staining of tissue sections, we
recommend the use of either the Santa Cruz Biotechnology, Inc. ABC Staining Systems or the ImmunoCruz™
Staining Systems. The ABC Staining Systems utilize
preformed avidin-biotinylated horseradish peroxidase
complex as a detection reagent, whereas the ImmunoCruz™ Staining Systems utilize a streptavidin-horseradish peroxidase complex. The ImmunoCruz™ Staining
Systems include all secondary reagents in a pre-diluted,
ready to use format. Complete research protocols are
included with all Staining Systems; brief protocols are
given below.
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All steps are carried out at room temperature in a humidified chamber. Allow all Staining System reagents
to reach room temperature prior to use. Tissue sections
should not be allowed to dry out at any time during
the procedure. Use suction to remove reagents after
each step, but avoid drying of specimens between
steps. Use sufficient reagents to cover the specimens
(approximately 100 µl per slide is usually adequate).
C. Formalin-Fixed, Paraffin-Embedded
Tissue Sections
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Fix tissue sections in formalin and embed in paraffin
blocks according to standard procedures.
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Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Or use pre-treated slides.
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Cut 4-6 micron thick tissue sections and apply to
slides. Deparaffinize in xylenes using three changes
for 5 minutes each. Hydrate sections gradually through
graded alcohols: wash in 100% ethanol twice for 10
minutes each, then 95% ethanol twice for 10 minutes
each. Wash in deionized H2O for 1 minute with stirring.
Aspirate excess liquid from slides.
Optional: Antigen unmasking may be performed at this
point. Certain antigenic determinants are masked by
formalin fixation and paraffin embedding and may be
exposed by one of several methods:
1) Heat treatment (recommended method): Place
slides in a container and cover with 10 mM sodium
citrate buffer, pH 6.0; or with 50 mM glycine-HCl
buffer (glycine: sc-29096), pH 3.5, with 0.01% (w/v)
EDTA (EDTA: sc-29092). Heat at 95° C for 5 minutes.
Top off with fresh buffer and heat at 95° C for 5
minutes (optimal incubation time may vary for each
tissue type). Allow slides to cool in the buffer for
approximately 20 minutes. Wash in deionized H2O
three times for 2 minutes each. Aspirate excess
liquid from slides.
2) Pepsin: Incubate sections for 10–20 minutes in
0.1% pepsin in 0.01 N HCl at room temperature.
Wash slides several times in deionized H2O. Aspirate
excess liquid from slides.
ABC STAINING SYSTEMS
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Incubate specimens for 1 hour in 1.5% normal blocking serum in PBS. Blocking serum ideally should be
derived from the same species in which the secondary
antibody is raised. Remove blocking serum from slides.
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Incubate with primary antibody for 30 minutes at room
temperature or overnight at 4° C. Optimal antibody
concentration should be determined by titration; recommended range is 0.5–5.0 µg/ml diluted in PBS with
1.5% normal blocking serum. Wash with three changes
of PBS for 5 minutes each.
䡲
Incubate for 30 minutes with biotin-conjugated secondary antibody as provided or at approximately 1 µg/ml
diluted in PBS with 1.5% normal blocking serum. Wash
3) Saponin: Incubate sections for 30 minutes in 0.05%
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䡲
䡲
䡲
with three changes of PBS for 5 minutes each.
䡲
Incubate for 30 minutes with avidin biotin enzyme
reagent. Wash with three changes of PBS for 5 minutes
each.
Prepare slides as described above for immunoperoxidase staining, omitting the final step involving treatment of cells with H2O2.
䡲
Use suction to remove reagents after each step, but
avoid drying of specimens between steps. Use sufficient reagent to cover the specimen (approximately
100–500 µl per slide is adequate).
䡲
Incubate specimens with 10% normal blocking serum
in PBS for 20 minutes to suppress non-specific binding
of IgG. Blocking serum ideally should be derived from
the same species in which the secondary antibody is
raised. Wash with PBS.
䡲
Incubate with primary antibody for 60 minutes. Optimal antibody concentration should be determined by
titration; recommended range is 0.5–5.0 µg/ml in PBS
with 1.5% normal blocking serum. Wash with three
changes of PBS for 5 minutes each.
䡲
Incubate for 45 minutes with either biotin-conjugated
or fluorochrome-conjugated secondary antibody diluted
to 1–5 µg/ml in PBS with 1.5%-3% normal blocking
serum. Optimal antibody concentration should be
determined by titration. Wash with three changes of
PBS. If fluorochrome-conjugated secondary antibody
is used, incubate in a dark chamber and omit the next
step.
䡲
Incubate with streptavidin-fluorescein for 15 minutes
in a dark chamber. Optimal streptavidin conjugate
concentration for a given application should be determined by titration; recommended range is 10–20 µg/
ml in PBS. Wash extensively with PBS.
䡲
Mount coverslip with aqueous mounting medium or
90% glycerol in PBS.
䡲
Examine using a fluorescence microscope with appropriate filters. Store slides in a dark location at room
temperature (UltraCruz™ Mounting Medium: sc-24941
or at 4° C (glycerol/PBS mount).
Incubate in peroxidase substrate as provided for 30
seconds to 10 minutes or until desired stain intensity
develops. Individual slides should be monitored to determine the proper development time. Wash sections
in deionized H2O for 5 minutes. If desired, counterstain
in Gill’s formulation #2 hematoxylin (sc-24973) for
5–10 seconds. Immediately wash with several changes
of deionized H2O.
Dehydrate through alcohols and xylenes as follows:
Soak in 95% ethanol twice for 10 seconds each, then
100% ethanol twice for 10 seconds each, then xylenes
three times for 10 seconds each. Wipe off excess xylene. Immediately add 1-2 drops of permanent mounting medium (e.g. Clarion sc-24942), cover with a glass
coverslip (sc-24975) and observe by light microscopy.
IMMUNOCRUZ™ STAINING SYSTEMS
䡲
Incubate specimens for 20 minutes in 1–3 drops of
serum block. Aspirate serum from slides.
䡲
Dilute primary antibody in serum block to 0.5–5.0 µg/
ml as determined by titration. Incubate for 2 hours.
Rinse with PBS then wash in PBS twice for 2 minutes
each on a stir plate. Aspirate excess liquid from slides.
䡲
Incubate for 30 minutes in 1–3 drops of biotinylated
secondary antibody. Wash as above.
䡲
Incubate for 30 minutes in 1–3 drops of HRP-streptavidin complex. Wash as above.
䡲
䡲
Add 1-3 drops HRP substrate mixture. Develop for 30
seconds to 10 minutes or until desired stain intensity
develops. Rinse with deionized H2O and transfer to a
deionized H2O wash for 2 minutes on a stir plate.
Counterstain, dehydrate and mount slides as described
under ABC Staining Systems.
7. IMMUNOFLUORESCENCE CELL STAINING
NOTE: For a listing of cover glasses and micro slides for
Immunohistochemistry, please see our catalog or visit our
website at www.scbt.com.
Santa Cruz Biotechnology, Inc.
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8. FLOW CYTOMETRY
A. Sample Preparation
Prepare cells according to cell type.
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BLOOD (HUMAN, MOUSE OR RAT)
䡲
䡲
䡲
䡲
For each 1 ml of blood, add 14 ml of room temperature
FCM Lysing solution (sc-3621) to lyse the red blood
cells. The cells will not lyse correctly if the solution is
cold.
Incubate for 5 minutes at room temperature on a rotator. Do not exceed 5 minutes, as the white blood cells
will begin to lyse beyond 5 minutes.
Centrifuge for 5 minutes at 1000 RPM for human blood,
2000 RPM for mouse or rat blood.
Carefully aspirate supernatant, then resuspend pellet
in approximately 50 ml cold 1X PBS. Take a small
sample to perform a cell count.
䡲
Centrifuge for 5 minutes at 1000 RPM for human blood,
2000 RPM for mouse or rat blood.
䡲
Aspirate supernatant.
䡲
Add approximately 5 ml of 0.2% EDTA (in PBS) to
plate. Using a Trypsin/EDTA solution in the place of
0.2% EDTA may compromise any cell surface staining.
䡲
Wait for cells to “round up.” Placing the cells in an incubator may speed up this process. Check the plate(s)
every 5 minutes.
䡲
Add approximately 5 ml of media to neutralize EDTA.
䡲
Pipette off cells, rinsing plate to ensure maximum recovery. Take a small sample to perform a cell count.
䡲
Centrifuge for 5 minutes at 1000 RPM.
䡲
Aspirate supernatant.
B. Cell Stimulation
Stimulate cells as necessary.
C. Stain Preparation
Fix cells or prepare live cells for staining.
MOUSE SPLEEN OR OTHER TISSUE
䡲
Harvest organ or tissue and prepare single cell suspension.
䡲
Pass cell suspension through a 70 µM cell strainer.
NOTE: It is very important to block Fc receptors for certain cell types including, but not limited to, mouse and
rat blood, mouse spleen, mouse bone marrow, etc. For
mouse or rat tissues, use sc-18867 L.
䡲
Centrifuge for 5 minutes at 1000 RPM.
LIVE STAINING
䡲
Discard supernatant and add 5 ml of room temperature
FCM Lysing solution (sc-3621).
䡲
Incubate for 2-3 minutes at room temperature, allowing larger pieces to fall to the bottom of the tube.
䡲
Carefully pipette the suspension out and deposit into
a clean tube. Take a small sample to perform a cell
count.
䡲
Centrifuge for 5 minutes at 1000 RPM.
䡲
Aspirate supernatant.
䡲
Once supernatant is aspirated from cell preparation,
resuspend pellet in enough 1X PBS to have a final cell
concentration of 10 million cells/ml.
䡲
Block by incubating the cell suspension with 1 mg of
sc-18867 L per 1 ml of cell suspension for 10 minutes.
Do not rinse. Proceed with staining.
FIXED AND PERMEABILIZED CELLS FOR INTRACELLULAR
STAINING
䡲
Once supernatant is aspirated from cell preparation,
resuspend pellet in enough 1X PBS to have a final cell
concentration of 10 million cells/ml.
SUSPENSION CELL LINE
䡲
Pipette off cells, rinsing plate to ensure maximum recovery. Take a small sample to perform a cell count.
䡲
Block by incubating the cell suspension with 1 µg of
sc-18867 L per 1 ml of cell suspension for 10 minutes.
䡲
Centrifuge for 5 minutes at 1000 RPM.
䡲
䡲
Aspirate supernatant.
Resuspend pellet in approximately 50 ml 1X PBS to
wash away any excess blocking antibody.
䡲
Centrifuge for 5 minutes at 1000 RPM.
䡲
Once supernatant is aspirated from cell preparation,
resuspend pellet in FCM Fixation Buffer (sc-3622).
Use mL per million cells.
MONOCLONAL/ADHERENT CELL LINE
䡲
Vacuum off media. Rinse plate once with 1X PBS.
Vacuum off PBS.
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䡲
䡲
䡲
䡲
䡲
䡲
Incubate for 30 minutes at room temperature on a
rotator.
DIRECT STAINING
Centrifuge for 5 minutes at 1500-2000 RPM. Cells get
more buoyant after fixation. If pellet is too small, spin
again at a higher RPM, but do not exceed 3000 RPM.
䡲
Label tubes.
䡲
Add 20 µl of fluorochrome-conjugated antibodies to
tubes.
䡲
Add 100 µl of the prepared cell suspension (equal to
1 million cells) to each tube.
䡲
Vortex and incubate for 15-30 minutes in a covered ice
bucket.
䡲
To wash off excess antibody following staining, add
1.5–2 ml of 1X PBS to each tube.
䡲
Centrifuge in tabletop microfuge for 5 minutes at 2000
RPM. This speed should be increased to 3000 or 4000
RPM for intracellular staining.
䡲
Aspirate supernatant, being careful not to disturb pellet.
䡲
Resuspend pellets in 500 µl of 1% paraformaldehyde.
Tubes can be stored in the dark for 24 hours (maximum
for intracellular staining) to 1 week (maximum for surface staining).
Pour off supernatant. Cells may be lost if aspirating
from this point on, so always decant. Use a quick
motion and don’t allow the supernatant to wash back
and forth over the cells.
Resuspend pellet in approximately 50 ml 1X PBS to
wash away any excess Fixation Buffer.
Centrifuge for 5 minutes at 1500–2000 RPM.
Decant supernatant. At this point, cells can be resuspended in a small amount of PBS and stored for up to
one month at 4° C. To permeabilize at this time, proceed to next step.
NOTE: You should only proceed with permeabilization
if you can stain immediately afterwards.
(WITH FLUOROCHROME -CONJUGATED ANTIBODIES)
䡲
If cells have been stored in PBS, centrifuge for 5 minutes at 1500-2000 RPM and decant supernatant.
䡲
Break up cell pellet and dropwise add the same amount
of COLD (stored at -20° C) FCM Permeabilization Buffer,
sc-3623 at 1 ml per 1 million cells. Vortex while adding.
䡲
Incubate for 5 minutes only at RT on a rotator.
䡲
Label tubes.
䡲
Immediately centrifuge for 5 minutes at 2000–2500
RPM. Cells are more buoyant after permeabilization
and much care must be excercised to maintain volume
of cells.
䡲
Add unconjugated primary antibodies to tubes. Use
approximately 1 µg per tube.
䡲
Add 100 µl of the prepared cell suspension (equal to
1 million cells) to each tube.
NOTE: Important: If a pellet is not recovered at this
step, be sure to spin again and try to recover more
cells.
䡲
Vortex and incubate for 15-30 min in a covered ice
bucket.
䡲
To wash off excess antibody following staining, add
1.5–2 ml of 1X PBS to each tube.
䡲
Centrifuge in tabletop microfuge for 5 minutes at 2000
RPM (or 3000–4000 RPM for intracellular staining).
䡲
Aspirate supernatant, being careful not to disturb pellet.
䡲
Add 100 ml of 1X PBS to each tube. Add fluorochrome-conjugated secondary antibodies to tubes.
Use 0.5–1 µg of antibody.
䡲
Vortex and incubate for 15–30 minutes in a covered ice
bucket.
䡲
Decant supernatant and add approximately 50 ml 1X
PBS to wash away any excess Permeabilization Buffer.
䡲
Centrifuge for 5 minuntes at 2000–2500 RPM.
䡲
Decant supernatant and resuspend pellet in enough
FCM Wash Buffer, sc-3624, for a final cell concentration of 10 million cells/ml. In the staining steps, use
FCM Wash Buffer in place of 1X PBS.
D. Staining
INDIRECT STAINING
(WITH FLUOROCHROME -UNCONJUGATED PRIMARY
ANTIBODIES AND FLUOROCHROME-CONJUGATED
SECONDARY ANTIBODIES)
Follow protocol for direct or indirect staining.
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䡲
To wash off excess antibody following staining, add
1.5–2 ml of 1X PBS to each tube.
䡲
Remove liquid in wells. Wash three times with PBS
containing 0.05% Tween-20 and dry plate.
䡲
Centrifuge in tabletop microfuge for 5 minutes at 2000
RPM (or 3000–4000 RPM for intracellular staining).
䡲
䡲
Aspirate supernatant, being careful not to disturb pellet.
Wash wells once with diethanolamine buffer (10 mM
diethanolamine, 0.5 mM MgCl2, pH 9.5) and remove
liquid.
䡲
Resuspend pellets in 500 µl of 1% paraformaldehyde.
Tubes can be stored in the dark for 24 hours (maximum
for intracellular staining) to 1 week (maximum for surface staining).
䡲
Dilute substrate (PNPP, sc-3720) in diethanolamine
buffer to a final concentration of 1 mg/ml. Add 50 µl/
well. Allow to develop for 10–20 minutes or until
positive control reaches an OD 405/490 of about 1.0.
Stop reaction by adding 50 µl of 0.1 M EDTA (EDTA:
sc-29092), pH 7.5. Read plates on microtiter plate
reader at OD 405/490.
E. Acquire
Acquire within 24 hours.
10. TRANSCRUZ™GEL SUPERSHIFT ASSAYS
9. ELISA ASSAYS
䡲
䡲
Coat microtiter plates with target protein diluted in
50 mM carbonate buffer at pH 9.0. Optimal concen
trations should be determined by titration, but for
purified antigens 50 µl per well at 1 µg/ml is usually
sufficient. Incubate overnight at 4° C covered with
parafilm.
Remove antigen solution. Add 200 µl/well of blocking
buffer (PBS containing 1% BSA and 0.02% azide) to
block non-specific protein binding. Incubate for 1–2
hours at room temperatureor overnight at 4° C.
䡲
Remove blocking buffer. Wash once with PBS with
0.02% azide. Damp strip wells or plates are usually
stable in resealable plastic storage bags for 4 weeks
at 4° C. Before using, remove excess liquid.
䡲
Add test antibody samples and controls at 50 µl/well
diluted in blocking buffer. Antibodies may be serially
diluted for determining titer or diluted to previously
determined working concentration for screening assays or antigen quantitation. Incubate 1 hour at room
temperature.
䡲
Wash plates three times with PBS containing 0.05%
Tween-20 (Tween-20: sc-29113), removing excess
liquid as above.
䡲
Add 50 µl/well of alkaline phosphatase-conjugated
secondary antibody diluted to 1:100–1:1000 in blocking buffer. Optimal antibody concentration is determined by titration. Incubate 1 hour at room
temperature.
Santa Cruz Biotechnology, Inc.
1.800.457.3801
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䡲
Label oligonucleotide probe with [γ P]-ATP to 50,000
cpm/ng by using polynucleotide kinase (for a listing of
these reagents and more, please see our catalog or
visit our website at www.scbt.com.).
䡲
Prepare gel shift reaction buffer as follows: 10 mM
Tris (Tris: sc-3715), pH 7.5, 50 mM NaCl, 1 mM dithiothreitol (DTT: sc-29089), 1 mM EDTA (EDTA: sc-29092),
5% glycerol (glycerol: sc-29095).
䡲
Prepare 20 µl reaction mixture containing 3–10 µg
nuclear extract and 1 µg poly dI-dC in gel shift reaction buffer. Add 0.5 ng labeled oligonucleotide probe
and incubate for 20 minutes at room temperature.
This constitutes the control sample for detection of
DNA-protein complexes.
䡲
To detect an antibody supershift or block of the DNAprotein complex, prepare reaction mixture as described
above, also adding 1–2 µl of the appropriate TransCruz™ Gel Supershift antibody per 20 µl of reaction
volume. Antibody is normally added subsequent to addition of labeled oligonucleotide probe, but result may
be improved by adding antibody prior to probe. Incubate
at 4° C for 1 hour to overnightor at room temperature
for 15–45 minutes.
䡲
Resolve DNA-protein complexes by electrophoresis
(25–35 ma) through a 4% polyacrylamide gel containing
50 mM Tris, pH 7.5, 0.38 M glycine (glycine: sc-29096)
and 2 mM EDTA. Dry the gel and visualize by autoradiography.
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fuge tube for the sonication step.
11. PEPTIDE NEUTRALIZATION
䡲
䡲
䡲
䡲
Blocking (neutralizing) peptides are available as negative controls for all Santa Cruz Biotechnology, Inc.
affinity-purified rabbit and goat polyclonal antibodies
and monoclonal antibodies raised against peptide
antigens. Antibody binding to antigen may be blocked/
competed by pre-absorption with the blocking peptide.
Determine the highest antibody dilution at which a
consistently positive result is achieved for the desired
test. For example, H-Ras (259) is recommended for immunoprecipitation at 1 µg/ml but is positive at a dilution
of 50 ng/ml.
䡲
Sonication conditions should be optimized since results may vary using different sonifiers. The following
conditions were established by using a Sonics VC130
with a 3 mm tip probe.
䡲
Sonicate on ice at power output setting = 5-6, continuous mode, 4 times at 30 second intervals.
䡲
Centrifuge extract for 15 minutes, 10,000 rpm at 4° C
and save supernatant (chromatin).
䡲
Determine protein concentration of supernatant.
䡲
For the IP step we recommend using 100–500 µg
protein and 0.1-1 µl TransCruz™ reagent (0.2-2 µg).
For blocking/competition, combine antibody (at a concentration determined by the aforementioned method)
with a five-fold (by weight) excess of blocking peptide
in a small volume (500 µl) of PBS. Incubate for up to
2 hours at room temperature or overnight at 4° C.
NOTE: Investigators may wish to consider using the
primary antibody conjugated to sepharose or magnetic
beads as an alternative to using secondary immunoprecipitation reagents (e.g. Protein A-Agarose) as described here. Combining primary antibodies directed
to different epitopes of the same protein may be advantageous in some cases.
Following blocking/competition, dilute antibody/peptide mixture into appropriate blocking buffer and proceed with the desired research application.
䡲
Preclear the chromatin solution by adding 50 µl Protein A/G PLUS-Agarose (sc-2003) and incubate for 30
minutes at 4º C. Centrifuge at full speed for 5 minutes
at 4º C.
䡲
Add primary antibody to the supernatant and incubate
overnight at 4° C.
䡲
Add 50 µl Protein A/G PLUS-Agarose (sc-2003) and
incubate for 2 hrs at 4º C.
䡲
Terminate cross-linking reactions by adding glycine to
a final concentration of 0.125 M.
Harvest beads by centrifugations at 12,000 rpm for 20
seconds and place tube in ice.
䡲
Pellet cells (2,000 rpm, 5 minutes) and wash once with
ice cold PBS.
Wash beads twice with 1 ml Lysis Buffer High Salt
(sc-45001).
䡲
Wash pellet four times with Wash Buffer (sc-45002).
䡲
Resuspend beads in 400 µl Elution Buffer (sc-45003).
䡲
Reverse cross-links by incubating tube in a 67º C water
bath, mixing occasionally over two hours. Remove
12. CHROMATIN IMMUNOPRECIPITATION
(ChIP) ASSAYS
NOTE: ChIP protocols vary widely. The following protocol
should be suitable for most experiments.
䡲
Wash cells twice with PBS at room temperature, resuspending to approximately 5x10 cells/ml (approximately 2x10 cells total). Add formaldehyde to a final
concentration of 1% and incubate at room temperature
for 10 minutes.
5
7
䡲
䡲
䡲
Resuspend cells in 6 ml Lysis Buffer (sc-45000) by
mixing gently.
䡲
Collect crude nuclear extract by microcentrifugation at
2,000 rpm, 5 minutes.
䡲
Wash again with PBS. Pellet may be frozen or processing may be continued as follows:
䡲
Resuspend pellet in approximately1.9 ml Lysis Buffer
High Salt (sc-45001) and transfer to 2 ml microcentri-
beads by centrifugation and continue incubating supernatant at 67º C overnight.
䡲
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Centrifuge for 3 minutes at 10,000 to remove any
residual beads and save supernatant.
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䡲
䡲
䡲
䡲
To isolate DNA, extract supernatant once with 500 µl
phenol/chloroform/isoamyl alcohol (25:24:1), vortex
thoroughly and separate phases by centrifuging tube
for 3 minutes at 14,000 rpm.
NOTE: Although highly efficient in a variety of cell
lines, siRNA Transfection Reagent: sc-29528 may not
be suitable for use with all cell lines.
DNA may be concentrated by using commercially
available kits.
䡲
Add the siRNA duplex solution (Solution A) directly
to the dilute Transfection Reagent (Solution B) using a
pipette. Mix gently by pipetting the solution and incubate the mixture 15–45 minutes at room temperature.
䡲
Wash the cells once with 2 ml of siRNA Transfection
Medium: sc-36868 Aspirate the medium and proceed
immediately to the next step.
䡲
For each transfection, add 0.8 ml siRNA Transfection
Medium (Solution A + Solution B) to each tube containing the siRNA: Transfection reagent mixture. Mix
gently and overlay the mixture onto the washed cells.
䡲
Incubate the cells 5–7 hours at 37° C in a CO2 incubator.
In a six well tissue culture plate, seed 2 x 10 cells per
well in 2 ml antibiotic-free normal growth medium
supplemented with FBS.
5
NOTE: This protocol is recommended for a well from
a 6 well tissue culture plate. Adjust cell and reagent
amounts proportionately for wells or dishes of different sizes.
NOTE: Longer transfection times may be desirable
depending on the cell line. However prolonged serum
starvation may result in unwanted cell detachment or
death.
Incubate the cells at 37° C in a CO2 incubator until
the cells are 60 - 80% confluent. This typically takes
18–24 hours.
NOTE: Healthy and subconfluent cells are required for
successful transfection experiments. It is recommended
to ensure cell viability one day prior to transfection.
䡲
NOTE: If a lower siRNA concentration is desired, dilute
siRNA appropriately with siRNA Dilution Buffer:
sc-29527.
Extract pooled aqueous phase with 600 µl chloroform/isoamyl alcohol.
NOTE: Santa Cruz Biotechnology, Inc. offers siRNA products for every human and mouse protein for which we
have a corresponding antibody. Most are listed throughout the catalog in the appropriate sections. For a complete list of all of the siRNA products we offer, please
see our web site at www.scbt.com.
䡲
NOTE: Optimal siRNA amount used for transfection
may vary for each target protein and should be determined experimentally.
Save the aqueous phase, back extract the organic
phase once with 100 µl 10 mM Tris, 1 mM EDTA,
pH 8.1 (TE) and pool aqueous phases.
13. siRNA MEDIATED INHIBITION OF GENE
EXPRESSION
䡲
NOTE: Do not add serum and antibiotics to the siRNA
Transfection Medium: sc-36868.
NOTE: Fluorescein Conjugated Control siRNA should
only be incubated for a total 5-7 hours at 37° C in a
CO2 incubator. At the end of incubation they are ready
to be assayed by fluorescent microscopy.
䡲
Add 1 ml of normal growth medium containing 2 times
the normal serum and antibiotics concentration (2x
normal growth medium) without removing the transfection mixture. If toxicity is a problem, remove the
transfection mixture and replace with 1x normal
growth medium.
䡲
Incubate the cells for an additional 18–24 hours.
䡲
Aspirate the medium and replace with fresh 1x normal
growth medium.
Prepare the following solutions:
Solution A: For each transfection, dilute 2–8 µl of
siRNA duplex (i.e. 0.25–1 µg or 20-80 pmols siRNA)
into 100 µl siRNA Transfection Medium: sc-36868.
Solution B: For each transfection, dilute 2–8 µl of
siRNA Transfection Reagent: sc-29528 into 100 µl
siRNA Transfection Medium: sc-36868. Peak activity
should be at about 6 µl siRNA Transfection Reagent.
Santa Cruz Biotechnology, Inc.
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䡲
Assay the cells using the appropriate protocol 24–72
hours after the addition of fresh medium in the step
above.
䡲
Incubate at 42° C for 2 minutes to anneal primer and
template.
䡲
Add 1 µl reverse transcriptase (200 units) and incubate
at 42° C for 50 minutes to extend the primer and then
terminate the reaction by incubating at 70° C for 15
minutes.
NOTE: Controls should always be included in siRNA
experiments. Use either Control siRNAs or Control
siRNA (Fluorescein Conjugates) (see table above). Each
contain a scrambled sequence that will not lead to the
specific degradation of any known cellular mRNA.
NOTE: For Western blot analysis prepare cell lysate
as follows: Wash cells once with PBS. Lyse cells in
300 µl 1x electrophoresis sample buffer (sc-24945:
Electrophoresis Sample Buffer, 2X) by gently rocking
the 6 well plate or by pipetting up and down. Sonicate
the lysate on ice if necessary.
NOTE: For RT-PCR analysis isolate RNA using the
method described by Chomczynski and Sacchi (1987.
Anal. Biochem. 162:156-159. Single-step method of
RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Chomczynski P, Sacchi N.) or a
commercially available RNA isolation kit.
14. SEMI-QUANTITATIVE NESTED RT-PCR
NOTE: Tm values for PCR primers offered by Santa Cruz
Biotechnology, Inc. range between 55-60 C (19-21 nt,
GC% ~55%). The A and B nested primer sets share
similar base pair length, GC% and Tm values.
NOTE: Nested PCR utilizes two pairs of PCR primers for a
single locus. The first primer pair A set amplifies within
the locus. The second primer pair B set (nested primers)
then binds within the “A” amplicon to produce a second
nested “B” amplicon.
NOTE: As an optional step add 1 µl RNase H (2 unit/µl)
and incubate at 37° C for 20 minutes.
2. First PCR Reaction
䡲
5 µl 10x PCR buffer (with or without* MgCl2)
5 µl 25 mM MgCl2
1 µl 10 mM dNTP
1 µl primer pair A
1 µl Taq DNA polymerase
2 µl cDNA and add water to 50 µl
* It may be necessary to vary the MgCl2 concentration, 2.5 mM final
concentration recommended.)
䡲
Incubate at 94° C for 2 minutes to denature the cDNA.
䡲
Perform 15–40 PCR cycles. Annealing and extension
conditions are primer and template dependent and
must be determined empirically for each templateprimer pair.
3. Second PCR Reaction
䡲
Prepare a solution containing:
* It may be necessary to vary the MgCl2 concentration, 2.5 mM final
concentration recommended.)
1 µl oligo (dT)12-18 (500 µg/ml)
1 ng-5 µg total RNA
1 µl 10 mM dNTPs
䡲
Incubate at 94° C for 2 minutes to denature the cDNA.
䡲
Perform 15–40 PCR cycles. Annealing and extension
conditions are primer and template dependent and
must be determined empirically for each templateprimer pair.
䡲
PCR products are separated on agarose gels and
visualized by ethidium bromide staining.
and add RNase-free water to a final volume of 12 µl
䡲
Incubate at 70° C for 5 minutes to minimize RNA
secondary structure, quick chill on ice and then add:
4 µl 5x reverse transcriptase buffer
2 µl 0.1 M DTT
1 µl RNase inhibitor
Santa Cruz Biotechnology, Inc.
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Prepare a solution containing:
5 µl 10x PCR buffer (with or without* MgCl2)
1 µl 10 mM dNTP
1 µl primer pair B
1 µl Taq DNA polymerase
1-5 µl first PCR product and add water to 50 µl
1. cDNA Synthesis
䡲
Prepare a solution containing:
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NOTE: Although highly efficient in a variety of cell
lines, shRNA Plasmid Transfection Reagent: sc108061 may not be suitable for use with all cell
lines.
15. shRNA PLASMID DNA MEDIATED
INHIBITION OF GENE EXPRESSION
䡲
In a six well tissue culture plate, grow cells to a
50-70% confluency in antibiotic-free normal growth
medium supplemented with FBS.
䡲
Add the shRNA Plasmid DNA solution (Solution A)
directly to the dilute shRNA Plasmid Transfection
Reagent (Solution B) using a pipette. Mix gently by
pipetting the solution up and down and incubate the
mixture 15-45 minutes at room temperature.
䡲
Wash the cells twice with 2 ml of shRNA Transfection
Medium: sc-108062 Aspirate the medium and proceed
immediately to the next step.
NOTE: This protocol is recommended for a well from
a 6 well tissue culture plate. Adjust cell and reagent
amounts proportionately for wells or dishes of different sizes.
NOTE: Healthy and subconfluent cells are required for
successful transfection experiments. It is recommended to ensure cell viability one day prior to
transfection.
䡲
NOTE: Do not use PBS as the residual phosphate may
compete with DNA and bind the shRNA Plasmid
Transfection Reagent, thereby reducing the transfection
efficiency.
Prepare the following solutions:
NOTE: The optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio should be determined
experimentally beginning with 1 µg of shRNA Plasmid
DNA and between 1.0 and 6.0 µl of shRNA Plasmid
Transfection Reagent as outlined below. Once the
optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio has been identified for a given
cell type, the appropriate amount of shRNA Plasmid
DNA/shRNA Plasmid Transfection Reagent complex
used per well should be tested to determine which
amount provides the highest level of transfection efficiency. For example, if the optimal shRNA Plasmid
DNA:shRNA Plasmid Transfection Reagent ratio is
1 µg:1 µl, then amounts ranging from 0.5 µg/0.5 µl
to 2.0 µg/2.0 µl should be tested.
Solution A: For each transfection, dilute 10 µl of resuspended shRNA Plasmid DNA (i.e. 1 µg shRNA Plasmid
DNA) into 90 µl shRNA Plasmid Transfection Medium:
sc-108062.
䡲
For each transfection, add 0.8 ml shRNA Plasmid
Transfection Medium to well.
䡲
Add the 200 µl shRNA Plasmid DNA/shRNA Plasmid
Transfection Reagent Complex (Solution A + Solution
B) dropwise to well, covering the entire layer.
䡲
Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution.
䡲
Incubate the cells 5-7 hours at 37° C in a CO2 incubator or under conditions normally used to culture the
cells. Longer transfection times may be desirable depending on the cell line.
䡲
Following incubation, add 1 ml of normal growth
medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium).
䡲
Incubate the cells for an additional 18-24 hours under
conditions normally used to culture the cells.
Solution B: For each transfection, dilute 1-6 µl of
shRNA Plasmid Transfection Reagent: sc-108061
with enough shRNA Plasmid Transfection Medium:
sc-108062 to bring final volume to 100 µl.
NOTE: Do not add antibiotics to the shRNA Plasmid
Transfection Medium: sc-108062.
NOTE: Optimal results may be achieved by using siliconized microcentrifuge tubes.
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OPTIONAL: For transient transfection, aspirate media
and replace with fresh1x normal growth medium.
Assay the cells using the appropriate protocol 24-72
hours after the addition of fresh medium in the previous step.
For selection of stably transfected cells, proceed with
puromycin selection as follows:
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NOTE: The working puromycin concentration for mammalian cell lines ranges from 1-10 µg/ml. Prior to
using the puromycin antibiotic (sc-108071), titrate the
selection agent to determine the optimal concentration for target cell line. Use the lowest concentration
that kills 100% of non-transfected cells in 3-5 days
from the start of puromycin selection.
48 hours post-transfection, aspirate the medium and
replace with fresh medium containing puromycin at
the appropriate concentration.
should be adjusted depending on the growth area of
the well or plate.
DAY 2
䡲
Prepare a mixture of complete medium with Polybrene® (sc-134220) at a final concentration of 5 µg/
ml.
䡲
Remove media from plate wells and replace with 1 ml
of this Polybrene/ media mixture per well (for 12-well
plate).
NOTE: Polybrene is a polycation that neutralizes
charge interactions to increase binding between the
pseudoviral capsid and the cellular membrane. The
optimal concentration of Polybrene depends on cell
type and may need to be empirically determined (usually in the range of 2-10 µg/ml). Excessive exposure to
Polybrene (> 12 hr) can be toxic to some cells.
Approximately every 2-3 days, aspirate and replace
with freshly prepared selective media.
NOTE: Controls should always be included in shRNA
experiments. Control shRNAs are available as 20 µg.
Each encode a scrambled shRNA sequence that will
not lead to the specific degradation of any known
cellular mRNA. Control shRNA Plasmids include:
sc-108060, sc-108065 and sc-108066.
NOTE: For Western blot analysis prepare cell lysate as
follows: Wash cells once with PBS. Lyse cells in 300 µl
1x Electrophoresis Sample Buffer (sc-24945) by gently
rocking the 6 well plate or by pipetting up and down.
Sonicate the lysate on ice if necessary.
䡲
Thaw lentiviral particles at room temperature and mix
gently before use.
䡲
Infect cells by adding the shRNA Lentiviral Particles to
the culture.
䡲
Swirl the plate gently to mix and incubate overnight.
The amount of viral particles to use varies greatly depending on the characteristics of the cell line used.
NOTE: For RT-PCR analysis isolate RNA using the
method described by P. Chomczynski and N. Sacchi
(1987. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159) or a commercially
available RNA isolation kit.
NOTE: Keep thawed shRNA Lentiviral Particles on ice.
Repeated freeze-thaw cycles and prolonged exposure
of the particles to ambient temperatures may result in
decreased viral titers.
NOTE: When transducing a shRNA lentiviral construct
into a cell for the first time we suggest using several
amounts of shRNA lentiviral particle stock. In addition,
we recommend to include one well with cells transduced with Control shRNA Lentiviral Particles (sc108080).
16: shRNA LENTIVIRAL PARTICLES
TRANSDUCTION
DAY 1
䡲
䡲
Plate target cells in a 12-well plate 24 hours prior to
viral infection.
Add 1 ml of complete optimal medium (with serum
and antibiotics) and incubate cells overnight. The cells
should be approximately 50% confluent on the day of
infection and (Day 2).
NOTE: It is possible to use other plate formats for
transduction as well. In this case, the amount of cells
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NOTE: Use copGFP Control Lentiviral Particles (sc108084) for measuring transduction efficiency.
DAY 3
䡲
Remove the culture medium and replace with 1 ml of
complete medium (without Polybrene).
䡲
Incubate the cells overnight.
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DAY 4
䡲
To select stable clones expressing the shRNA, split
cells 1:3 to 1:5, depending on the cell type, and continue incubating for 24-48 hours in complete medium.
DAY 5-6 and forward
䡲
䡲
䡲
Select stable clones expressing the shRNA via
Puromycin dihydrochloride (sc-108071) selection.
For puromycin selection, use an amount sufficient to
kill the non-transduced cells. Puromycin concentrations
ranging from 2 to 10 &mico;g/ml are usually sufficient,
but a puromycin tritation is recommended when using
a new cell line.
17. GENERAL SOLUTIONS
NOTE: For a listing of reagents required to prepare the
following solutions, please see our catalog or visit our
website at www.scbt.com.
䡲
Blotto A (for general use): 1x TBS, 5% milk, 0.05%
Tween-20. Available pre-made (sc-2333).
䡲
Blotto B (for use with anti-phosphotyrosine antibodies):
1x TBS, 1% milk, 1% BSA, 0.05% Tween-20. In some
cases, milk may be left out entirely, but this will result
in somewhat higher backgrounds. Available pre-made
(sc-2335). For all phospho-specific antibodies, add
0.01% (v/v) of each Phosphatase Inhibitor Cocktail A
and B (sc-45044 and sc-45045) to inhibit phosphatase
activity.
䡲
Diaminobenzidine tetrahydro-chloride (DAB): Dissolve
5 mg DAB in 100 ml 100 mM Tris-HCl, pH 7.6 and add
0.1 ml 0.3% hydrogen peroxide. Prepare fresh DAB
solution daily.
䡲
Electrophoresis sample buffer (2X): 100mM 2-(N-Morpholino)- ethanesulfonic acid(MES), 10 mM Na EDTA,
15% glycerol, 1.5 %SDS, 0.3% Triton X, 25mM TCEPHCL, 7.5 mM DTT, 0.0025% Bromophenol Blue. Available pre-made (sc-24945).
䡲
Phosphate buffered saline (1x PBS): 9.1 mM dibasic
sodium phosphate, 1.7 mM monobasic sodium phosphate and 150 mM NaCl. Adjust pH to 7.4 with NaOH.
Available pre-made in liquid (sc-24946) and powder
(sc-24947) forms.
䡲
RIPA buffer: 1x PBS, 1% Nonidet P-40 or Igepal CA-630,
0.5% sodium deoxycholate, 0.1% SDS. This may be
made in large volumes. Add protease inhibitors at
time of use from the following stock solutions. Also
available pre-made (sc-24948).
Replace medium with fresh puromycin-containing
medium every 3-4 days, until resistant colonies can be
identified. Pick several colonies, expand them and
assay them for stable shRNA expression.
NOTE: Resulting puromycin-resistant clones may have
varying levels of shRNA expression due to the random
integration of the lentiviral construct into the genome
of the cell.
NOTE: For shRNA expression analysis by Western
Blot, prepare cell lysate as follows:
䡲
Wash cells once with PBS.
䡲
Lyse cells in 100 µl of a 1:1 mixture of 2x Electrophoresis Sample Buffer (sc-24945) and RIPA Lysis Buffer
(sc-24948) by gently rocking the 12-well plate or by
pipetting up and down.
䡲
inactivate after transduction and integration of shRNA
constructs into genomic DNA of target cells.
Sonicate the lysate on ice if necessary.
NOTE: For shRNA expression analysis by RT-PCR, isolate RNA using the method described by P. Chomczynski and N. Sacchi (1987. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162: 156-159)
or a commercially available RNA isolation kit.
BIO SAFETY
Lentiviral particles can be employed in standard Biosafety Level 2 tissue culture facilities (and should be
treated with the same level of caution as with any other
potentially infectious reagent). Lentiviral particles are
replication-incompetent and are designed to self-
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1) 10 mg/ml PMSF (sc-3597) in isopropanol (add at
10 µl/ml RIPA)
2) Aprotinin (sc-3595) (add at 50 KIU/ml RIPA)
3) 100 mM sodium orthovanadate (sc-3540) in frozen
aliquots (add at 10 µl/ml RIPA)
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䡲
Subbing solution: 0.3% (w/v) gelatin, 0.05% chromium
potassium sulfate in distilled H2O.
䡲
Tris buffered saline (1x TBS): 10 mM Tris-HCl, pH 7.4;
150 mM NaCl (Tris: sc-3715). Available pre-made in
liquid (sc-24951) form.
Santa Cruz Biotechnology, Inc.
1.800.457.3801
831.457.3800
fax 831.457.3801
Europe
+00800 4573 8000
49 6221 4503 0
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