Western blot protocol

Procedure for western blotting

Solutions and reagents:

Lysis buffers

These buffers may be stored at 4°C for several weeks or for up to a year aliquoted and stored at -20°C.

RIPA buffer (Sigma #R0278)
Complete Protease Inhibitor cocktail (Roche #)

PhosSTOP Phosphatase inhibitor Cocktail (Roche #)

1xTBS buffer
20 mM Tris-HCl

150 mM NaCl

Milli-Q Water

pH 7.5

1xTBST buffer

1xTBS buffer

0.1% (v/v) Tween-20

pH 7.5

Running Buffer

100 ml 10x Tris/Glycine/SDS (Bio-Rad #161-0732)

900 ml Milli-Q water

Transfer Buffer

100 ml 10x Tris/Glycine (Bio-Rad #161-0734)

200 ml methanol

700 ml Milli-Q water

Blocking buffer

5% (w/v) milk

Add to TBS buffer. Mix well.

Antibody dilution buffer
5% (w/v) milk

Add to TBST buffer. Mix well.

Procedure:

1. Sample lysis

Preparation of lysate from cell culture

1.     Place the cell culture dish in ice and wash the cells with ice-cold PBS.

2.     Aspirate the PBS, then add ice-cold lysis buffer (1 ml per 10⁷ cells/100 mm dish/150 cm²   flask; 0.5ml per 5x10⁶ cells / 60 mm dish / 75 cm² flask).

3.     Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube.

4.     Maintain constant agitation for 30 minutes at 4°C.

5.     Sonicate for 1 minutes (2 seconds working and 3 seconds rest for each cycle)

6.     Spin at 16,000 x g for 20 minutes in a 4°C pre-cooled centrifuge.

7.     Gently remove the tube from the centrifuge and place on ice. Transfer the supernatant to a fresh tube kept on ice, and discard the pellet.

2. Sample preparation

1.     Determine the protein concentration for each cell lysate. (Appendix 1)

2.     To the remaining volume of cell lysate, adjust the concentration of cell lysate at 1 ug/ul with lysis buffer, 4xSample Buffer (Invitrogen # NP0007) and 10xSample Reducing Agent (Invitrogen # NP0009).

3.     To reduce and denature: Boil each cell lysate in sample buffer at 100°C for 5 minutes and aliquot. Store lysates at -20°C. Note: aliquot cell lysates (50- 100 μl) to avoid repeat freeze/thaw cycles.

4.     Defrost tubes containing cell lysate at 37°C. Centrifuge at 16,000 x g in a microcentrifuge for 5 minutes.

3. Loading and running the gel

1.     Load equal amounts of protein into the wells of the SDS-PAGE gel (Bio-Rad # 567-1085), along with molecular weight markers. Load 10- 40 μg of total protein from cell lysate.

2.     Run the gel for 1 to 2 hours at 100 V.

4. Transferring the protein from the gel to the membrane

1. Cut off nick top left-hand corner of resolving gel for orientation.
2. Measure the dimensions of the gel and note the positions of the ladder bands.
3. Transfer gel, while still attached to glass plate, to box containing TGM and peel off gently with a spatula.
4.  Agitate 15-20 seconds at RT to remove salts and SDS.
5. Cut a piece of PVDF membrane to the size of the gel and mark and/or clip one corner as the top left-hand corner. Handle only with flat forceps.
6. Immerse membrane in Transfer buffer for 10-15 seconds.
7. Cut 2 pieces of >3mm filter paper to the dimensions of the gel (or slightly bigger).
8. Open a gel holder cassette in a casserole dish, black side down and hinges to the left and below the black side.
9. Soak a fiber pad with Transfer buffer and place in the center of the black side.
10. Soak one piece of filter paper with Transfer buffer and place on top of the fiber pad.
11. Roll out bubbles with glass tube and add 3ml of Transfer buffer onto the top.
12. Place gel on top of filter paper.
13. Roll out bubbles with glass tube and add 3ml of Transfer buffer onto the top.
14. Place membrane on top of gel, with the gel’s top left mark facing the membrane’s top left mark.
15. Roll out bubbles with glass tube and add 3ml of Transfer buffer onto the top.
16. Soak a second piece of filter paper with Transfer buffer and place on top of the membrane.
17. Roll out bubbles with glass tube and add 3ml of Transfer buffer onto the top.
18. Soak a second piece of fiber pad and place on top of stack.
19. Roll out bubbles with glass tube and add 3ml of Transfer buffer onto the top.
20. Close the gel holder cassette.
21. Place in a transfer tank (orient the red and black sides of the cassette with the red and black panels of the electrode) and fill with Transfer buffer (~2000ml).
22. Place the tank in a styrofoam box containing ice.
23. Run 75 minutes at 100V.

5. Antibody staining

1.     Block the membrane for 1-2 hours at room temperature using 5% blocking solution.

2.     Incubate membrane with appropriate dilutions of primary antibody (Appendix 2) in antibody dilution buffer overnight at 4°C.

3.     Wash the membrane in three washes of 1xTBST, 5 minutes each.

4.     Incubate the membrane with the recommended dilution of labeled secondary antibody in antibody dilution buffer at room temperature for 1 hour.

5.     Wash the membrane in three washes of 1xTBST, 5 minutes each, then rinse in 1xTBS.

6.     Scan the membrane according to the manual of Obyssey.

Appendix 1

Preparation of Standards and Working Reagent

A. Preparation of Diluted Albumin (BSA) Standards

B. Preparation of the BCA Working Reagent (WR)

1. Use the following formula to determine the total volume of WR required:

(# standards + # unknowns) × (# replicates) × (volume of WR per sample) = total volume WR required

Example: for the standard test-tube procedure with 3 unknowns and 2 replicates of each sample:

(9 standards + 3 unknowns) × (2 replicates) × (2mL) = 48mL WR required

Note: 2.0mL of the WR is required for each sample in the test-tube procedure, while only 200 μl of WR reagent is required for each sample in the microplate procedure.

2. Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B). For the above example, combine 50mL of Reagent A with 1mL of Reagent B.

Note: When Reagent B is first added to Reagent A, turbidity is observed that quickly disappears upon mixing to yield a clear, green WR. Prepare sufficient volume of WR based on the number of samples to be assayed. The WR is stable for several days when stored in a closed container at room temperature (RT).

Microplate Procedure (Sample to WR ratio = 1:8)

1. Pipette 25μL of each standard or unknown sample replicate into a microplate well (working range = 20-2000μg/mL).

Note: If sample size is limited, 10μL of each unknown sample and standard can be used (sample to WR ratio = 1:20). However, the working range of the assay in this case will be limited to 125-2000μg/mL.

2. Add 200μL of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds.

3. Cover plate and incubate at 37°C for 30 minutes.

4. Cool plate to RT. Measure the absorbance at or near 562nm on a plate reader.