P0 (P-zero)
Catalog Number:
Product Type:
Immunogen Sequence:
Applications:
Data Sheet
CH23009
Host:
Affinity Purified
Peptide corresponded to a region of the Po
gene product shared between the mouse
(NP_032649, NCBI) and human
(NP_000521, NCBI) gene products.
Species
Reactivity:
Format:
Chicken
Human, Mouse
Liquid PBS (pH 7.2; 10 mM;
isotonic 0.9%, w/v) with sodium
azide (0.02%, w/v). Antibody
Concentration : 200 ug/ml
Immunocytochemistry: 1:100 -1:200
Immunohistochemistry: 1:100 -1:200
Western blot: 1:250 -1:500
Dilutions listed as a recommendation. Optimal dilution should be determined by investigator.
Storage:
References:
Store at 4°C in the dark. Under these conditions, the antibody should have a shelf life of at
least 12 months (provided they remain sterile). Do not freeze the antibody unless you want t o
store it for longer periods of time. Note, however, that each time an antibody preparation is
frozen, about half its binding activity is lost.
Wiebke Kallenborn-Gerhardt, Katrin Schröder, Domenico Del Turco, Ruirui Lu, Katharina
Kynast, Judith Kosowski, Ellen Niederberger, Ajay M. Shah, Ralf P. Brandes, Gerd
Geisslinger, and Achim Schmidtko. NADPH Oxidase-4 Maintains Neuropathic Pain after
Peripheral Nerve Injury. The Journal of Neuroscience, 25 July 2012, 32(30): 10136-10145;
doi: 10.1523/JNEUROSCI.6227-11.2012
Silvia Murillo-Cuesta, Guadalupe Camarero, Águeda González-Rodríguez, Lourdes
Rodríguez-de la Rosa, Deborah J Burks, Carlos Avendaño,Ángela M Valverde and Isabel
Varela-Nieto1. Insulin Receptor Substrate 2 (IRS2)-Deficient Mice Show Sensorineural
Hearing Loss That Is Delayed by Concomitant Protein Tyrosine Phosphatase 1B (PTP1B)
Loss of Function. Online address: http://www.molmed.org. doi: 10.2119/molmed.2011.00328
Christopher P Irmen, Sandra M Siegel and Patrick A Carr. Localization of SSeCKS in
unmyelinated primary sensory neurons. Journal of Brachial Plexus and Peripheral Nerve Injury
2008, 3:8doi:10.1186/1749-7221-3-8.
Application Notes
Immunostaining Cell Cultures
1. Draw of culture medium with aspirator and add 1 ml of 3.7 % formalin in PBS solution to the dish. (make up from 10mls
Fisher 37% formalin plus 90mls PBS, the Fisher formalin contains 37% formaldehyde plus about 1% methanol which may be
relevant sometimes). Let sit at room temp for 1 minute. (can add 0.1% Tween 20 to PBS used here and all subsequent steps
to reduce background; probably best not to do this first time round though as it may extract your antigen or help wash your
cells off the dish).
2. Take off the formalin/PBS and add 1ml of cold methanol (-20°C, kept in well sealed bottle in fridge). Let sit for no more
than 1 minute.
3. Take off methanol and add 1ml of PBS, not letting the specimen dry out. To block nonspecific antibody binding can add
~10ml (=1%) of goat serum (Sigma), and can incubate for 30 minutes. Can then add antibody reagents. Typically 100ml of
hybridoma tissue culture supernatent or 1ml of mouse ascites fluid or crude serum. Incubate for 1 hour at room temp. (or
FOR RESEARCH USE ONLY
NEUROMICS’ REAGENTS ARE FOR IN VITRO AND CERTAIN NON-HUMAN IN VIVO EXPERIMENTAL USE ONLY AND NOT INTENDED FOR USE IN ANY HUMAN
CLINICAL INVESTIGATION, DIAGNOSIS, PROGNOSIS, OR TREATMENT. THE ABOVE ANALYSES ARE MERELY TYPICAL GUIDES. THEY ARE NOT TO BE CONSTRUED
AS BEING SPECIFICATIONS. ALL OF THE ABOVE INFORMATION IS, TO THE BEST OF OUR KNOWLEDGE, TRUE AND ACCURATE. HOWEVER, SINCE THE
CONDITIONS OF USE ARE BEYOND OUR CONTROL, ALL RECOMMENDATIONS OR SUGGESTIONS ARE MADE WITHOUT GUARANTEE, EXPRESS OR IMPLIED, ON OUR
PART. WE DISCLAIM ALL LIABILITY IN CONNECTION WITH THE USE OF THE INFORMATION CONTAINED HEREIN OR OTHERWISE, AND ALL SUCH RISKS ARE
ASSUMED BY THE USER. WE FURTHER EXPRESSLY DISCLAIM ALL WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. V3-07/2012
www.neuromics.com
Neuromics Antibodies • 5325 West 74th Street, Suite 8 • Edina, MN 55439
phone 866-350-1500 • fax 612-677-3976 • e-mail [email protected]
can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight, exact time not too critical). Can do very gentle shaking for
well adherent cell lines (3T3, Hek293 etc.).
4. Remove primary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give
three washes in PBS.
5. Add 0.5 mls of secondary antibody. These are fluorescently labeled Goat anti chicken antibodies and are conjugated to
ALEXA dyes and are from Molecular probes (Eugene Oregon, the ALEXA dyes are sulphonated rhodamine compounds and
are much more stable to UV than FITC, TRITC, Texas red etc.). Typically make 1:2,000 dilutions of these secondaries in
PBS plus 1% goat serum, BSA or non-fat milk carrier. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to
1 hour, or can do 4°C overnight). Can do gentle shaking for well adherent cell lines (3T3, HEK293 etc.).
6. Remove secondary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give
three washes in PBS.
7. Drop on one drop of Fisher mounting medium onto dish and apply 22mm square coverslip. View in the microscope!
Immunostaining Tissue
Solutions
PBS - sodium phosphate-buffered (100 mM; pH 7.2) isotonic (0.9% NaCl, w/v) saline Antibody dilution buffer (PBS with
0.1% non-ionic detergent, such as Triton X-100 or Tween-20). For anti-fading, use Neuromics’ i-BRITE Plus –Catalog#:
SF40000 or make your own fluorescein anti-fading reagent -- Make up a 2 mg/ml phenylene diamine solution in PBS
(phenylene diamine requires extensive vortexing to put it into solution). Once the phenylene diamine is completely dissolved,
O
add an equal volume of glycerol and mix. This reagent will last about a week at -20 C. Discard this reagent when it starts to
turn dark brown.
Other Reagents
Fluorescein-labeled goat anti-chicken IgY
1. Prepare your tissue sections or cultured cells as you normally would. Wash your sections or cells for 1 min with PBS at
room temperature.
2. Incubate your sections or cells with your chicken primary antibodies (diluted in "antibody dilution buffer") for at least 1 hour
at room temperature. The concentration of your antibody may be anywhere from 1:50-1:150 depending on the titre of the
antibody and the concentration of your antigen.
3. Wash your sections or cells over a 10 minute period at room temperature (with two changes of PBS).
4. Incubate your sections or cells with fluorescein-labeled goat anti-chicken IgY (1:500 dilution in "antibody dilution buffer” for
1 hour at room temperature. Be sure to keep these slides or culture dishes in subdued light (e.g., in a drawer) to avoid
bleaching of the fluorescein dye.
5. Repeat step #4
6. Add a drop of "fluorescence anti-fading reagent" (i-BRITE Plus) to your sections or cells. Place a coverslip over the
section. If you want to reduce messiness, you may also seal the coverslip by painting the edges with nail polish.
7. Store the slides or culture dishes in the refrigerator (in the dark).
Western Blotting
Solutions
PBS – sodium phosphate-buffered (100 mM; pH 7.2) isotonic (0.9% NaCl, w/v) saline
Antibody dilution buffer (PBS with 0.1% non-ionic detergent, such as Triton X-100 or Tween-20)
Blocking Reagent
Other Reagents
Horseradish Peroxidase-labeled goat anti-chicken IgY (Catalog#: CH23104)
SuperSignal® Substrate for Western Blotting (Pierce Chemical Company, Catalog #34080)
Steps
1. Run your SDS-polyacrylamide Laemelli gel and blot the protein bands onto nitrocellulose membrane, following standard
methods (see chapter 12 of Harlow and Lane, 1988). Be sure NOT to use PVDF or other nylon-based membranes, as
chicken antibodies tend to bind non-specifically, contributing to higher backgrounds.
2. Block non-specific sites on your membrane by incubating the membrane for 30 min at room temperature with Chicken
Blocking Reagent using gentle agitation.
3. Wash the blot three times in PBS with 0.05% Tween-20 at room temperature. Each wash should be at least 10 minutes
with gentle agitation.
4. Incubate your blot for at least 1 hour at room temperature with your chicken antibody diluted in “antibody dilution buffer.”
Optimal dilutions of chicken antibody may be as low as 1:500 or as high as 1:50,000 depending on the immunogenicity of
the antigen. Generally, the longer the period of incubation, the better (provided that the blot doesn’t dry out) — even
overnight incubations are fine.
5. Wash the blot in PBS, as described above (step 3).
FOR RESEARCH USE ONLY
NEUROMICS’ REAGENTS ARE FOR IN VITRO AND CERTAIN NON-HUMAN IN VIVO EXPERIMENTAL USE ONLY AND NOT INTENDED FOR USE IN ANY HUMAN
CLINICAL INVESTIGATION, DIAGNOSIS, PROGNOSIS, OR TREATMENT. THE ABOVE ANALYSES ARE MERELY TYPICAL GUIDES. THEY ARE NOT TO BE CONSTRUED
AS BEING SPECIFICATIONS. ALL OF THE ABOVE INFORMATION IS, TO THE BEST OF OUR KNOWLEDGE, TRUE AND ACCURATE. HOWEVER, SINCE THE
CONDITIONS OF USE ARE BEYOND OUR CONTROL, ALL RECOMMENDATIONS OR SUGGESTIONS ARE MADE WITHOUT GUARANTEE, EXPRESS OR IMPLIED, ON OUR
PART. WE DISCLAIM ALL LIABILITY IN CONNECTION WITH THE USE OF THE INFORMATION CONTAINED HEREIN OR OTHERWISE, AND ALL SUCH RISKS ARE
ASSUMED BY THE USER. WE FURTHER EXPRESSLY DISCLAIM ALL WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. V3-07/2012
www.neuromics.com
Neuromics Antibodies • 5325 West 74th Street, Suite 8 • Edina, MN 55439
phone 866-350-1500 • fax 612-677-3976 • e-mail [email protected]
6. Incubate your blot for 1 hour at room temperature with a 1:5,000 to 1:10,000 dilution (in “antibody dilution buffer”) of
horseradish peroxidase-labeled goat-anti-chicken IgY with gentle agitation.
7. Wash the blot in PBS, as described above (step 3).
8. Develop the blot for 5-10 minutes at room temperature using SuperSignal® HRP Substrate Working Solution.
9. Place the blot against X-ray film and exposure from 10 seconds to 5 minutes (depending on the intensity of the signal).
10. Develop the X-ray film as you normally would.
NOTE: Chicken antibodies tend to display higher backgrounds on polyvinylidene fluoride (PVDF) and other nylon-based
membranes. For this reason, we recommend using nitrocellulose membranes for western blotting applications.
Images: Po, P-zero or MPZ and PMP22 expression in the
sciatic nerve of WT and Nox4−/− mice after SNI. A,
Western blot analysis of the myelin-specific proteins MPZ
and PMP22 in the day 14 SNI sciatic nerve (proximal
nerve stump) and the uninjured control sciatic nerve.
GAPDH was used as loading control. Note that MPZ and
PMP22 protein expression is significantly decreased after
SNI in WT mice but not in Nox4−/− mice. n = 3 mice per
group. Data are presented as mean ± SEM (*p < 0.05). B,
Immunostaining of the day 14 SNI sciatic nerve shows
increased MPZ immunoreactivity in Nox4−/− mice
compared with WT mice, whereas immunoreactivity of the
neuronal marker NF200 is similar in both genotypes.
Scale bar, 10 μm.
FOR RESEARCH USE ONLY
NEUROMICS’ REAGENTS ARE FOR IN VITRO AND CERTAIN NON-HUMAN IN VIVO EXPERIMENTAL USE ONLY AND NOT INTENDED FOR USE IN ANY HUMAN
CLINICAL INVESTIGATION, DIAGNOSIS, PROGNOSIS, OR TREATMENT. THE ABOVE ANALYSES ARE MERELY TYPICAL GUIDES. THEY ARE NOT TO BE CONSTRUED
AS BEING SPECIFICATIONS. ALL OF THE ABOVE INFORMATION IS, TO THE BEST OF OUR KNOWLEDGE, TRUE AND ACCURATE. HOWEVER, SINCE THE
CONDITIONS OF USE ARE BEYOND OUR CONTROL, ALL RECOMMENDATIONS OR SUGGESTIONS ARE MADE WITHOUT GUARANTEE, EXPRESS OR IMPLIED, ON OUR
PART. WE DISCLAIM ALL LIABILITY IN CONNECTION WITH THE USE OF THE INFORMATION CONTAINED HEREIN OR OTHERWISE, AND ALL SUCH RISKS ARE
ASSUMED BY THE USER. WE FURTHER EXPRESSLY DISCLAIM ALL WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. V3-07/2012
www.neuromics.com
Neuromics Antibodies • 5325 West 74th Street, Suite 8 • Edina, MN 55439
phone 866-350-1500 • fax 612-677-3976 • e-mail [email protected]