General Protocol for Western Blotting
ProtocolBulletin 6376

Key Solutions and Reagents

Blocking buffer

Lysis buffer: Radioimmunoprecipitation
assay buffer (RIPA buffer)

3% bovine serum albumin (BSA) in TBST

50 mM Tris-HCl, pH 8.0
150 mM NaCl
1% Nonidet P-40 (NP-40) or 0.1% Triton X-100
0.5% sodium deoxycholate
0.1% sodium dodecyl sulphate (SDS)

Stripping buffer

20 ml 10% SDS
12.5 ml 0.5 M Tris HCl, pH 6.8
67.5 ml ultrapure water
0.8 ml 2-mercaptoethanol

1 mM sodium orthovanadate

Procedure

1 mM NaFSample prep (based on a typical cell culture scenario)

Protease inhibitors tablet (Roche)

1. 	Place the cell culture dish in ice and wash the cells with
ice-cold Tris-buffered saline (TBS).

Loading buffer: 2x Laemmli buffer

4% SDS
10% 2-mercaptoethanol

2.	Aspirate the TBS, then add ice-cold RIPA buffer (1 ml per
100 mm dish).

0.004% bromophenol blue

3.	Scrape adherent cells off the dish using a cold plastic cell
scraper and gently transfer the cell suspension into a
precooled microcentrifuge tube.

0.125 M Tris-HCl4. Maintain constant agitation for 30 min at 4°C.

Check the pH and adjust to pH 6.8 if necessary.

5.	If necessary, sonicate 3 times for 10–15 sec to complete
cell lysis and shear DNA to reduce sample viscosity.

20% glycerol

Running buffer: Tris/Glycine/SDS

25 mM Tris
190 mM glycine
0.1% SDS
Transfer buffer

25 mM Tris
190 mM glycine
20% methanol
For proteins larger than 80 kD, we recommend thatSDS be included at a final concentration of 0.1%.

6.	Spin at 16,000 x g for 20 min in a 4°C precooled centrifuge.
7.	Gently remove the centrifuge tube and place it on ice.
Transfer the supernatant to a fresh tube, also kept on ice,and discard the pellet.
8.	Remove a small volume (10–20 μl) of lysate to perform
a protein assay. Determine the protein concentration
foreach cell lysate.
9.	If necessary, aliquot the protein samples for long-term
storage at –20oC. Repeated freeze and thaw cycles cause
protein degradation and should be avoided.

Ponceau S staining buffer

10.	Take 20 μg of each sample and add an equal volumeof 2x Laemmli sample buffer.

0.2% (w/v) Ponceau S

11.	Boil each cell lysate in sample buffer at 95°C for 5 min.

5% glacial acetic acid12.	Centrifuge at 16,000 x g in a microcentrifuge for 1 min.

Tris-buffered saline with Tween 20 (TBST) buffer

20 mM Tris, pH 7.5
150 mM NaCl
0.1% Tween 20

General Protocol for Western Blotting

Protein separation by gel electrophoresis

Imaging and data analysis

1.	Load equal amounts of protein (20 μg) into the wells ofa mini (8.6 x 6.7 cm) or midi (13.3 x 8.7 cm) format SDSPAGE gel, along with molecular weight markers.

1.	Apply the chemiluminescent substrate to the blot
according to the manufacturer’s recommendation.

2.	Run the gel for 5 min at 50 V.
3.	Increase the voltage to 100–150 V to finish the runin about 1 hr.
Gel percentage selection depends on the size of the protein ofinterest. A 4–20% gradient gel separates proteins of all sizes
very well. For details, please refer to the Protein Blotting Guide,bulletin 2895.
Transferring the protein from the gel to the membrane

1. Place the gel in 1x transfer buffer for 10–15 min.
2.	Assemble the transfer sandwich and make sure no air
bubbles are trapped in the sandwich. The blot should
be on the cathode and the gel on the anode.
3.	Place the cassette in the transfer tank and place an ice
block in the tank.
4.	Transfer overnight in a coldroom at a constant currentof 10 mA.	Note: Transfer can also be done at 100 V for 30 min–2 hr, but the methodneeds to be optimized for proteins of different sizes.

2.	Capture the chemiluminescent signals using a CCD
camera-based imager.	Note: The use of film is not recommended in this step because of its
limited dynamic range.

3.	Use image analysis software to read the band intensityof the target proteins.
Stripping and reprobing

1. Warm the buffer to 50°C.
2.	Add the buffer to the membrane in a container designatedfor stripping. Incubate at 50°C for up to 45 min withsome agitation.
3. Rinse the blot under running water for 1 hr.
4.	Transfer the membrane to a clean container, wash
5 times for 5 min with TBST.
5. Block in 3% BSA in TBST at room temperature for 1 hr.
6.	Incubate overnight in the primary antibody solution(against the loading control protein) at 4°C.
 ote: The antibody should be diluted in the blocking buffer at theN
manufacturer’s recommended ratio.

Antibody incubation

7. Rinse the blot 3–5 times for 5 min with TBST.

1.	Briefly rinse the blot in water and stain it with Ponceau S
solution to check the transfer quality.

8.	Incubate in the HRP-conjugated secondary antibody
solution for 1 hr at room temperature.

2.	Rinse off the Ponceau S stain with three washeswith TBST.

9. Rinse the blot 3–5 times for 5 min with TBST.

3.	Block in 3% BSA in TBST at room temperature for 1 hr.
4.	Incubate overnight in the primary antibody solution against
the target protein at 4°C.	Note: The antibody should be diluted in the blocking buffer according to
the manufacturer’s recommended ratio. Primary antibody may be applied
to the blot for 1–3 hr at room temperature depending on antibody quality
and performance.

5.	Rinse the blot 3–5 times for 5 min with TBST.

Note: The antibody can be diluted using 5% skim milk in TBST.

Imaging and data analysis

1.	Apply the chemiluminescent substrate to the blot following
the manufacturer’s suggestions.
2.	Capture the chemiluminescent signals using a CCD
camera-based imager.	Note: The use of film is not recommended in this step because of its
limited dynamic range.

6.	Incubate in the HRP-conjugated secondary antibody
solution for 1 hr at room temperature.

3.	Use image analysis software to read the band intensityof the loading control proteins.

Note: The antibody can be diluted using 5% skim milk in TBST.

4.	Use the loading control protein levels to normalize thetarget protein levels.

7. Rinse the blot 3–5 times for 5 min with TBST.

Nonidet is a trademark of Shell International Petroleum Co. Triton is a trademarkof Dow Chemical Company. Tween is a trademark of ICI Americas Inc.

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Bulletin 6376 Ver C

US/EG

17-0657 0517

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