WESTERN BLOT PROTOCOL

M.Fero 12/2011

(Includes sample preparation, Bradford Assay, SDS-PAGE, semi-dry transfer, antibody staining and ECL development.)

Materials: See below.

Harvesting cells:   

1. Prepare lysis buffer by adding fresh protease inhibitors (100x stocks listed below) to RIPA or TG buffer, plus 1/1000 vol. of PMSF (from a 200 mM stock).  Also add fresh phosphase inhibitors (from 100x stock) if kinase assays will be done. RIPA buffer is preferred, but it may be too harsh for some proteins. TG buffer, in contrast, may not lyse cells or nuclei as effectively, but it may be better for preserving cyclin D catalytic activity.
2. Scrape or trypsinize cells in culture. Spin and resuspend 10⁷ cells in 150 µL lysis buffer. For tissues, dounce 100 mg. of tissue in 1 mL cell lysis buffer.
3. Sonicate sample on ice to fragment the genomic DNA. Be careful not to let samples overheat while sonicating (the tubes should remain cool to touch).
4. Spin at maximum speed, 4ºC to remove debris.  Transfer supernatant to a fresh tube. 
5. 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.
6. Prior to use, the protein content of samples should be assayed.

Protein Quantitation:
Comments
The chief goal is to determine the relative protein abundance across samples to ensure equivalent loading of samples on the gel.  A second goal is to ensure that the absolute quantity of protein loaded is in a range that is sufficient for visualization but does not exceed the capacity of the gel.

If an abundant complex mixture is being assayed (e.g. cell or tissue extracts) then an A280 measurement may suffice.  However, A280 measurements are dependent on the amino acid composition of the sample, chiefly tryptophan and tyrosine, so A280 readings may not be appropriate for adjusting the concentrations of two different purified proteins or two different tissue types. A280 readings are sensitive to the presence of contaminating nucleic acids (DNA and RNA). 

The Bradford assay is relatively easy, sensitive, and less dependent on amino acid composition. It has a limited linear range, so it should be repeated on samples that give high readings and are subsequently diluted. It is recommended to measure all samples, that will be run on a single gel, together in a single Bradford assay, since there may be day to day variability in the results.

Bradford assay:    

• Dilute  Bradford reagent to 1/5x by adding 1 vol. reagent + 4 vol. H₂O.  
• Aliquot 0.8 mL diluted Bradford reagent into 1.5 mL tubes
• Add 2 µL of protein extract and mix by vortexing.  Incubate 2-5 min. at room temp.
• As a negative control add 2 µL of your protein lysis buffer to a separate 0.8 mL aliquot of Bradford reagent.
• Zero the spectrophotometer at 600 nm using the negative control.
• Measure A₆₀₀ for each sample in the same order that they were prepared (since the Bradford reagent absorbance will gradually increase over time).
• 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 simultaneously.  If necessary more accurate values may be obtained by adjusting the results according to a standard curve of a known protein. 
  

   SDS PAGE (Volumes used for 1 mm BioRad Mini-Protean gel system)

	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	-	-	-	-
H2O	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.)

7. Use 4 mL of resolving buffer for a 1mm MiniProtean gel. Gently overlay resolving buffer with ethanol to shield the buffer from air which will inhibit polymerization. Rinse off ethanol with water when gel has polymerized, and drain out the water. 
   
8. Overlay with stack gel and place a 10 or 15-well comb.
9. Prepare 10 - 50 µg protein in 15 - 25 µL of lysis buffer per lane (the volume depends on comb size). Add 1/4 vol of 5x SDS loading buffer to each sample. Heat on a 95°C block x 3 min prior to loading, then hold on ice.
   
10. Load samples into wells, along with lanes dedicated to a prestained MW marker and (+) and (-) controls.
11. 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):   

12. Cut 15 Whatman 3M filter sheets to 5.5 x 8.5 cm and one PDVF membrane by the same dimensions.
13. Soak filter paper in buffers A (6 sheets), B (3 sheets), and C (6 sheets).
14. Wet PDVF membrane in methanol. Hydrate in H₂O,then equilibrate in buffer B.
15. 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):

16. Invert this sandwhich onto the base (+) eletrode of the transfer apparatus. Remove saran wrap. Place (-) electrode on top of sandwich.
    
17. Transfer for 1 hr. at 40 mA per blot for 1 mm thick gels.  (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, e.g. use 160 mA if blotting for gels simultaneously.  The running time should be proportional to the thickness of the gel, so this may be reduced to 45 min. for 0.75 mm gels, or increased to 90 min. for 1.5 mm gels. High MW proteins (> 200 kD), or high percentage acrylamide gels may require longer transfer times. 
    

Antibody staining:   

17. 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.  Meanwhile, stain the residual proteins in the gel, with Coumassie blue (see below).
18. 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. Alternatively, if an appropriate fluorescently-labeled secondary antibody was used, then the blot may be scanned on an Odyssey imager (Li-Cor).
  

Coumassie blue staining:
Note: Following electrotransfer a small amount of protein (~10% of the total) will remain in the gel, assuming a 1mm gel was blotted for the time listed above. If a thinner gel is used then only a small amount of high MW material may remain or else the electrotransfer time may be proportionally reduced. Having a small amount of residual protein in the gel is convenient because it can be post-stained with Coumassie blue in order to document consistent protein loading across samples. Alternatively, membranes may be stained with Ponceau S solution (Sigma) for the same purpose.

19. When the gel is removed from the transfer stack (in step 13) rinse it briefly in H2O and place it in a staining tray. Gently agigate the gel in Coumassie Stain for 30 min.
    
20. Rinse in a small volume of Destain and then cover in Destain in a tray and gently aggitate for one hour. Repeat 1-2 times as necessary. Keep a balled up Kimwipe in the tray while destaining in order to absorb stain particles and maintain clarity of the destain solution. 
    
21. Photograph the gel on a white background for documentation. 
    
22. After use return the Coumassie stain to a glass bottle as it can be reused multiple times. The Destain solutions should be discarded with organic wastes. 

Interpretation of Results
Western blots are usually not quantified and thus are limited to qualitative interpretations, e.g. "The amount of protein X is higher in sample 1 than in sample 2", or "The levels of protein Y is unchanged across the samples". Still, some investigators choose to immunostain blots a 2nd time with an antibody against a protein, such as actin or tubulin, which are expressed at stable levels, as a "protein loading control". This gives visual reassuance that the changes seen for the protein of interest was not due to technical problems with protein loading. Regardless, of whether such a control is used, researchers should establish that both the experimental protein (and the loading control) are expressed within the "linear range" of the assay. For example, ECL may exhibit a threshold effect wherein the reduction of the target protein below a certain level is associated with a disappearance of a band rather than a reduction of its intensity.  The detection system may also become "saturated" for proteins with high expression levels, such that significant differences in levels will appear to be the same.  This is often the case for actin loading controls if a high concentration of antibody is used.

Quantitation
Western blots can be quantified with a reasonable level of accuracy if one is careful about the technical setup. A traditional way to do this is to scan films and perform densitometry. Fluorescent antibodies can also be used, in conjunction with an Odyssey imager, and exhibit a greater dynamic range and accuracy than densitometry. In either case, a standard curve comprised of two-fold serial dilutions of a positive control should be run in parallel to verify the linearity of the assay and to quantify the results. Minor differences in protein loading may be normalized by the differences seen in the actin or tubulin internal control, assuming that this too exhibits a linear relationship in a standard curve.

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 at -20°C.
Aprotinin 10 mg/mL (1000x) Store at -20°C.
PMSF: Phenylmethylsulfonyl flouride, 200mM in ethanol (100x) . 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. to 7.5 mL with H2O	-
7.5 mL Glycerol	10% (v/v)

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 g SDS (electrophoresis grade)	0.1% (w/v)
q.s. to 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.

Non-fat dried milk.