WESTERN BLOT PROTOCOL

M.Fero 3/04

Harvesting cells:

1) Add fresh protease inhibitors (100x stocks listed below) to RIPA or TG lysis buffer
plus 1/1000 vol of PMSF (200 mM stock). Also add fresh phosphase inhibitors (100x stock listed below) if kinase assays will be done.

2) Scrape or trypsinize cells in culture. Spin and resuspend 10⁷ cells in 150uL lysis buffer. For tissues, dounce 100 mg. of tissue in 1 mL cell lysis buffer.

3) Sonicate on ice to fragment the genomic DNA. Spin at maximum speed, 4ºC to remove debris. Transfer supernatant to a fresh tube. To retain kinase activity it is important to not allow the samles to freeze solid. Add 1 vol. glycerol (final = 50% v/v). The glycerol is viscous so, to facilitate pipetting, you should first cut the ends off of the pipetman tips with a pair of scissors. Mix thoroughly by a combination of pipetting and vortexing. Keep the extracts on ice when in use. Otherwise store the extracts at -20ºC. Ice crystals will form if the glycerol was not well mixed.

4) Quantitate protein by measuring A₂₈₀ or run Bradford assay:

Aliquot 0.8 mL of 1/4x Bradford reagent (diluted in H₂0) into 1.5 mL tubes
Add 2 µL of protein extract and mix by vortexing. Incubate 2 min. room temp.
Use 2 µL of lysis buffer to one tube with 1/4x Bradford reagent as a negative control to zero machine.
Measure A₆₀₀ for each sample.
To calculate the approximate protein concentration (µg/µL) multiply A₆₀₀ x 10. In order to ensure relative precision it is best if all of the extracts to be run on a single gel are quantified at the same time. If necessary more accurate values may be obtained by adjusting the results according to a standard curve of a known protein. For most purposes only the relative precision of samples (and their controls) is important.

SDS PAGE (Volume for 1 mm BioRad MiniProtean gel)

	Stack Gel (4 mL)	Separating gel (10 mL)
Acrylamide concentration	-	5%	10%	12%	15%
MW Range (kDal):	-	60 - 200	16 - 70	14 - 60	12 - 45
30% Acrylamide mix (29:1 acrylamide:bis-acrylamide)	0.67 mL	1.7 mL	3.3 mL	4 mL	5 mL
1.5M Tris pH8.8	-	2.5 mL	2.5 mL	2.5 mL	2.5 mL
1M Tris pH6.8	0.5 mL	-	-	-	-
H20	2.4 mL	5.7 mL	4.1 mL	3.4 mL	2.4 mL
10% SDS	40 µL	100 µL	100 µL	100 µL	100 µL
10% ammonium persulfate	30 µL	50 µL	50 µL	50 µL	50 µL
TEMED	3 µL	5 µL	5 µL	5 µL	5 µL

(Tris buffers must be made from Tris-base, and are pH'd with conc. HCl. Store acrylamide, 10% APS, and TEMED at 4ºC.)

1) Use 4 mL of resolving buffer for a 1mm MiniProtean gel. Gently overlay with ethanol. Rinse with H₂O when polymerized. Overlay with stack gel using a 10 or 15 well comb.

2) Use 10 - 50 µg protein in 15 - 25 µL of lysis buffer per lane (depending on comb size) Add 1/4 vol of 5x SDS loading buffer. Heat on 95°C block x 3 min prior to loading and store on ice. Load wells along with a prestained MW marker and (+) and (-) controls.

3) Run at 200 v. for 1 hr or until dye front runs off the bottom of the gel. Thicker gels (1.5 mm) will run hotter and should be nearly submerged in running buffer or run at lower voltages.

Electrotransfer (using Ellard Instruments HEB 2020 semi-dry blotter):
1) Cut 15 Whatman 3M filter sheets to 5.5 x 8.5 cm and one PDVF membrane by the same dimensions.
2) Soak filter paper in buffers A (6 sheets), B (3 sheets), and C (6 sheets).
2) Wet PDVF membrane in methanol. Hydrate in H₂O, then equilibrate in buffer B.
3) Separate glass plates. Rinse gel in H₂O. Discard stack. Create transfer sandwich on Saran wrap. (Soak filters in the appropriate solutions for 2 min. and squeeze out the extra solution):

Sandwich from bottom to top:
Saran wrap
Buffer A filters (6 sheets)
Gel
PVDF (prewetted in B).
Buffer B filters (3 sheets)
Buffer C filters (6 sheets)

4) Invert this sandwhich onto the base (+) eletrode of the transfer apparatus. Remove saran wrap. Place (-) electrode on top of sandwich. Transfer for 1 hr. at 40 mA per blot. (The protein will migrate out of the gel towards the (+) electrode and will stick to the PVDF membrane). Note: The necessary current is a function of the surface area of the gel sandwich so the mA must be increased proportional to the number of gels being run. One hour is sufficient to run the majority of proteins out of a 1 mm thick gel. Thicker gels or high MW proteins (> 200 kD) may require longer transfer times.

Antibody Staining:
1) When transfer is complete: Remove filter paper sandwich from electro-blotter apparatus. Mark MW bands with ink from a ball point pen (Papermate ink won't wash out). For orientation, nick the corner above MW markers. Stain gel, with gentle agitation, in Coumassie blue x15 min. Save the stain for reuse. Then gently agitate the gel in destain x 2 hrs with a crumpled KimWipe to help absorb dye. Plave the stained gel on a white surface and photograph under white lights to document the consistency of protein loading.

2) Stain the PDVF membrane with antibody as follows (after each step rinse well in several changes of TNT x 10 min):

5% Milk in 0.5% TNT (2.5 gm milk in 50 mL TNT) x 30-60 min. Rinse in TNT.
Primary antibody, 8 mL x 1 hr.(most are 1/1000 in 0.5% TNT). Rinse in TNT.
Secondary antibody 8 mL (1:10,000 HRP-anti-rabbit IgG) Rinse in well in TNT
Add ECL mix (1:1 mix of Amersham reagents 1 and 2) and immediately expose to film x 1 min.

MATERIALS:

RIPA cell lysis buffer	TG cell lysis buffer
10 mM NaPO4, pH7.2	20 mM HEPES, pH7.2
0.3 M NaCl	1% Triton-X
0.1% SDS	10% glycerol
1% NP40
1% DOC (deoxycholate)
2 mM EDTA

Protease Inhibitors:
Leupeptin 10 mg/mL (1000x, store -20°C otherwise keep on ice)
Aprotinin 10 mg/mL (1000x, store -20°C otherwise keep on ice)
PMSF: (phenylmethylsulfonyl flouride, 100x) 200mM in ethanol. Store at 4°C.

100x Phosphatase inhibitors (for kinase assays)
100 mM NaF
50 mM NaVanadate
800 mM ß-glycerol phosphate

5x Laemmli sample buffer (15 mL)	1x Concentrations
1.5 gm SDS	2% (w/v)
3.75 mL 1M Tris, pH 6.8	50 mM
0.015 gm bromphenol blue	0.2 mg/mL
1.16 gm DTT	0.1 M DTT
q.s. 7.5 mL H₂O	10% (v/v)
7.5 mL Glycerol

10x SDS Running Buffer (8L)	1x Concentration
1440 g Glycine (75 g/mole)	250 mM
242 g Tris-Base (121 g/mole)	25 mM
80 gm SDS (electrophoresis grade)	0.1% (w/v)
q.s. 8 L with H₂O

Western Transfer Solutions
Solution A: 25 mM Tris Base, 20% v/v isopropanol, 40 mM e-aminocaproic acid.
Solution B: 25 mM Tris Base, 20% v/v isopropanol.
Solution C: 250 mM Tris Base, 20% v/v isopropanol.

Coomassie Stain and Destain:
Coomassie Stain: 0.25% w/v brilliant blue (Sigma B-0770), 50% v/v methanol, 7.5% v/v glacial acetic acid. Filter through Whatman #1.
Destain: 10% (v/v) methanol, 10% (v/v) glacial acetic acid.

0.5% TNT: 0.5% Tween-20, 0.15 M NaCl, 25 mM Tris pH 7.4.

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