Coomassie and Western Blot on PVDF

PVDF Coomassie Staining -> Western Blot

This method enables you to combine Coomassie staining and a Western Blot without having to run 2 gels.

Briefly: In normal Coomassie staining, the destaining/staining process fixes the proteins into the gel, making transfer impossible. This is why you cannot run a Western on a stained gel, even if the gel has been destained.

In this protocol, transfer occurs immediately after running the SDS gel. With some modifications, the PVDF (or nitrocellulose) membrane can be stained as one would typically stain a Coomassie gel. The membrane can then be destained fully and a normal Western blot can be performed with the membrane. Adapted/modified from this paper.

NOTES: This method will lose pigment In the Coomassie staining step. PVDF does not retain dye as well as a normal SDS gel during the destaining process and has a higher background.

As a result, protein bands that would appear very dark in a normal Coomassie-stained gel will be slightly lighter on your PVDF membrane. The Western blot/antibody staining will not be affected by this method-- staining will be just as effective. If you need extremely high-resolution bands on your gel, I would recommend running a separate gel.

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Materials

Destain Solution ("Stain 1 solution")

• 40% H2O
• 50% ethanol
• 10% acetic acid + ADDITIONAL ACETIC ACID to speed up destaining process-- up to 25%

Staining Solution ("Stain 2 Solution")

• 87.5% H2O
• 5% EtOH
• 7.5% acetic acid
• .25% Coomassie dye (G250)

TBST

• TBS .05% tween20

Blocking Buffer

• TBST
• 5% milk powder

Antibodies

• Primary: 1:5000 in blocking buffer of purified mouse anti-HIS In 4C (NOT FITC, which Is for fluorescence). There are normally aliquots of this frozen In the fridge, 50 mL
• Secondary: 1:5000 peroxidase anti mouse IgG in -20 antibody box

Procedure

1. Run an SDS PAGE gel as normal. It shouldn't matter if it is the stained or unstained kind-- I've only used the stained-- but you can use either. Just make sure you use a ladder.

2. Once your gel Is done running, rinse the gel as you would to prepare it for a Western in diH2O. Perform WB as normal: transfer protein onto PVDF membrane using Turbo blot method (7 minutes for mini gel).

Our gels our TGX mini-gel. You could also use the TGX process, which is shorter, and also in the turbo category on the machine, but I use the mini-gel 7 min one.

3. After transfer, rinse the PVDF membrane with diH2O for a few minutes to remove residual buffer.

4. Incubate PVDF membrane in staining solution (Solution 2 labeled on bench). Should contain .25% Coomassie dye, normal parameters.

DO NOT MICROWAVE! This will damage the membrane.

5. The PVDF membrane will absorb the dye faster than the bands will stain. At 10 minutes, the membrane will likely be completely blue and will have absorbed all the dye In the solution. Give it a few more minutes-- I usually let it stain for 30-60 min.

You will likely NOT be able to visualize the bands in the membrane since the background is so high. This is fine, the destain step is very effective and quick.

6. Once the membrane has been rocking with dye for at least 30 minutes, begin the destaining process. Pour off the stain solution and add the destain solution (Solution 1, labeled on the bench as Destain). Replace the destaining solution as it becomes saturated with dye-- usually, I replace it once or twice.

To speed up the destaining process, pour in additional acetic acid. The paper this method Is based on uses around 25% acetic acid-- our destain solution is around 10%, so you can increase. If you choose to increase the concentration of acetic acid (I always do), remember that higher concentrations of acetic acid can damage your protein.

7. Let the membrane destain until the bands are visible and the background has decreased, then rinse the membrane with diH2O or TBST. Depending on the amount of additional acetic acid, it will take around 5-10 minutes to destain the background.

8. Once satisfied with the amount of background destaining, remove the destain solution and rinse/place the membrane in diH2O.

Do NOT leave the membrane in destain solution while imaging. The destain solution is very efficient and your membrane will be leaking dye and lose pigment as you are imaging.

9. Image the membrane-- Blot -> Colorimetric (no filter). You can lay the membrane directly on the Imager without damaging it.

10. Pour water/TBST off the membrane and add "stain 1 solution" and rock to continue destaining the membrane so that the protein bands are no longer visible, thereby preparing it for the Western Blot.

You can add acetic acid add this step to increase destaining speed. Replace the destaining solution as it becomes saturated with dye.

10. Check the membrane after around 5 minutes. The destaining process with additional acetic acid is rapid and will likely occur in under 10 minutes. Be careful not to over-destain and damage the protein. Your membrane should have your ladder visible and no other blue bands.

If everything is gone except for a few very light bands that were likely just a lot of protein, It's fine. These bands will destain throughout the blotting process-- just make sure 95% of everything is destained from the gel before proceeding with the WB. If you do not destain enough, you will have Coomassie background when you image your blot and your results will not be accurate.

11. Ensure that you wash your PVDF membrane with TBST to remove any staining buffer before blocking. Wash for 5 min, 3x with rocking.

12. Block in milk buffer for 1 hr -- proceed with Western as normal.