Whole Cell Extraction from Yeast Cells Using Denaturing
Buffer
To yeast cell pellet in 1.5 ml tube add 2 volumes of acid washed beads (100 to 500
ul volume of beads).
Add a volume of extraction buffer equal to approximately twice that of the cell/bead
volume (200 to 600 ul of buffer), I never use more than 600 ul of buffer.
Vortex for a full 3 minutes on your best vortex, use a timer.
Spin for 10 minutes at 4°C. The cell debris will be at the bottom, then the beads,
then the clear supernatant. Collect the clear supernatant to a fresh tube; then put
the pipet tip into the beads and collect additional supernatant but not deep enough
to get the cell debris.
Do a Bradford diluting samples as needed.
These are not stable for freeze thaw so take an aliquot of known concentration and
add Sample Loading Buffer and keep at -20°C and keep the remainder at -80°C
and limit freeze thaw cycles.
This is Becky’s version of the protocol from YeasTract Protein Extraction Kit from
Zygam Inc.
Extraction Buffer
9 M Urea
5 mM EDTA
54.05 g
1 ml 0.5M EDTA
water to 100 ml
Beads - 0.5 mm from Research Products International #9831
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Bradford Assay
Bradford reagent – BioRad protein assay dye reagent #500-0006
Make a fresh BSA dilution: 5 ul of 10 mg/ml BSA into 95 ul of 0.15 M NaCl
Make a Blank and 4 standards with BSA in microfuge tubes:
0.15 M NaCl Protein Ex.
Buffer
Blank
95
5
Standard #1
90
5
#2
85
5
#3
80
5
#4
75
5
Unknowns
95
BSA
0.5 mg/ml
0
5
10
15
20
5 ul of sample
Add 95 ul of 0.15 M NaCl to microfuge tubes and add 5 ul of each sample.(This can
be altered according to concentration.)
Add 1ml of diluted Bradford reagent (1:5) and vortex briefly; allow to stand for 10
minutes at room temperature.
Turn on visible light on spectrophotometer and set OD to 595nm. Pipet 800ul of
each sample to disposable cuvette. Read.
Use standard readings to plot a scatter graph in Excel. Add a linear trend line, and
have the equation and R-squared value diaplayed on the chart. Check R-squared
value to see if any standards should be dropped. Use the equation to calculate the
values of the unknowns. Divide the value by 5 to get ug/ul.
4 X Sample Loading Buffer
Add 100 ul of 4 M DTT to 900 ul of 4.5 X Sample Loading Buffer Stock. We use SLB
with very high DTT concentration due to the nature of our capsid proteins.
4.5 X Sample loading buffer
11.2 ml
8 ml
3.6 g
1 M Tris pH6.8
water
SDS
Dissolve SDS in water and Tris solution;
add to 18 ml of glycerol in 50 ml culture
tube; add 900 ul of 2% Bromophenol Blue.
Mix well and make 900 ul aliquots (-20°C)
4M DTT
3.1 g DTT to 5 ml final volume of water; make 200 ul aliquots. (-20°C)
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Western Gel, Transfer and Blot
Gel – 4-20% gradient gel from Pierce #252xx in Tris-HEPES buffer
Bio-Rad Mini-PROTEAN gel apparatus
Add sample buffer to samples and heat at 75ºC for at least 5 min.(or Boil)
Insert precast gel into cassette fill with 1 x running buffer, blow the wells.
Run gels at 100 to 120 Volts until BφB is to the green gasket or near the bottom.
Transfer Using Semi-Dry – BioRad Trans-Blot Semi-Dry Transfer Cell #170-3940
Use discontinuous buffer system:
Put 0.1% SDS in the cathode buffer (upper), this helps elute the proteins from the
acrylamide gel.
Put 15% Methanol in the anode buffer (lower), this helps the proteins bind to the
PVDF membrane.
Per Bio-Rad Tech note 2134 Anthony K. Tan
CAPS transfer buffer is recommended over glycine buffer for proteins with a pI over
8.5 and since our gag pI 8.411, CA pI 6.36, NC pI 11.23, IN pI 8.78 and pol pI
9.04. We use CAPS transfer buffer.
5X Stock Transfer Buffer
36.34 g Tris base
44.26 g CAPS
Water to 1 liter
Working buffers:
Anode buffer
20 ml 5 X Stock
15 ml MetOH
65 ml water
Cathode buffer
20 ml 5 x Stock buffer
1 ml of 10% SDS
79 ml water
The membrane should be wetted in 100% Methanol and then soaked in Anode
buffer for at least 30 min.
The gel should be equilibrated in Cathode buffer for 15 min.(0.75 mm thickness)
For proteins less than 10K daltons do quick rinses rather than soaking.
Transfer for mini gels is 15 to 30 minutes at 10-15 Volts. For large gels (or
multiple mini gels) 15-25 Volts for 30 to 60 minutes. Do not exceed 25 Volts!
A Current limit of 3 mA/cm2 for large gels and 5.5 mA/cm2 for mini gels should not
be exceeded. We have been using 12 Volts for 45 min for one or two mini-gels.
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Blot – Immobilion-P PVDF membrane, Millipore #IPVH00010
ECL Plus Western Blotting Detection, GE Healthcare #RPN2133
Use either PBS + T or TBS + T as prefered. Have buffer with 2.5 to 5% milk ready
and place membrane directly into this for blocking. Block on rocker for 1 hr at RT.
Add primary anti-body to milk solution. (ie: 1 ul anti-CA to 20 ml of milk solution
{1:20,000}) and incubate on rocker for 1 hr at RT.
Rinse and then wash 2-3 times for 10 min. at RT
Add fresh milk buffer with secondary antibody (ie: 1 ul of anti-rabbit in 20 ml of
milk {1:20,000}) and incubate 1 hr at RT on rocker.
Rinse and then wash 2-3 times for 10 min. at RT
Combine ECL reagents at 1:40 and react with membrane on either glass plate or
Saran wrap. Drain ECL reagent, wrap membrane in Saran wrap and off to the Dark
Room.
OR
Take ECL aliquots along with blot and Saran wrap to the Fuji imager and when you
determine the machine is free react mixed ECL reagents with blot and expose.
1 liter of TBS:
20 ml
37.5 ml
qs to 1 L
1 M Tris pH 7.4
4 M NaCl
Water
20 mM
150 mM
For 1xTBS + 0.1% Tween (TBS+T), add 5 ml of 20% Tween
5 x PBS: for 1 liter
56.78g
13.8 g
5.8 g
Na2HPO4 anhydrous
NaH2PO4.H2O
NaCl
80 mM
20 mM
100 mM
For 1xPBS + 0.1% Tween (PBS+T), add 5 ml of 20% Tween to 1 liter
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