Absence of the protein of interest on the membrane after transfer could be due to either poor transfer or the protein is crossing the membrane due to larger pore size. In your case, it seems that the protein of interest is crossing through PVDF due to larger pore size/increased voltage or time of transfer. Since your protein of interest is of low molecular weight, larger pore size (0.45 um) followed by increased voltage or time of transfer could be the issue since markers and control samples run in parallel are giving the right signal.

I would suggest the followings:

I) Since your protein is of almost 20 kDa, try to use 0.22 um) pore size PVDF (larger pore size PVDF may not hold the transferred protein and it may cross the membrane easily).

II) Reduce the voltage and increase the time of transfer

III) Reduction of voltage will minimize the heat generation that normally increase the resistance and destabilize the transfer buffer.

Reducing the voltage will further allow the protein of your interest to stuck on the 0-22um pore size membrane.

It is normally seen that if the gel is stained post-transfer, it will show the band of protein of your interest, as no transfer system is 100% efficient.

Good luck

@Shail K Chaube

Hello Shail, thank you very much for your reply.

I think it makes sense so I should try a new transfer with a smaller pore membrane (0.22 um).

But, I still have a doubt, why did the recombinant protein known here in the laboratory of the same size used as a "positive control" appear on the membrane? She is even smaller. Furthermore, on the polyacrylamide gel that we used in the transfer and stained with comassie, it does not appear, indicating that it has been fully transferred. Unlike this protein that we are trying to characterize, produced in Pichia pastoris, which did not appear in the membrane and remained entirely in the polyacrylamide gel. We have already tried an overnight transfer and with a lower voltage. Would you have any suggestions? Thank you very much for all your considerations. Att, Samara

Hi Samara Rocha,

As I suggested the membrane and few other issues that might be associated with your problem.

Since the problem exist with the poor transfer of the protein of your interest. I would ask some more clarification:

1. Did you check with Ponceau stain that the protein of your interest has been transferred on the PVDF membrane? If yes, then you may have to check your primary as well as secondary antibody issues.

2. If protein has not been transferred on the PVDF membrane and it is almost stuck on the SDS-PAGE, then its your binding to rechecked the entire procedure and fix the issue in order to get the signal on the membrane.

Rechecked if you have followed the followings:

1. Cast a 15% SDS-PAGE and run the gel slowly at 90V with a LMW marker and stop the gel run before the dye front runs out of gel (hope you might have done it).

2. Prepare your transfer buffer as per your transfer system (Semi-dry or Wet).

3. Prepare transfer buffer fresh to avoid methanol evaporation.

4. Keep the transfer buffer chilled condition by putting it in ice box one hour before its use for transfer

5. Keep the whole transfer unit at cold place in order to prevent heat denaturation of proteins during transfer.

6) Keep the PVDF membrane for 5-10mins in undiluted Methanol.

7) Carefully arrange gel as well as blotting membrane ensuring that there are no bubbles by repeatedly rolling over the stack with an empty falcon/glass rod.

8) Run the transfer at 110V for 80 mins for best transfers.

After rechecking the above steps, if you are still not getting the band of your interest, then recheck your antibody quality, expiry, dilutions, exposure time etc.

I am sure after going through all these steps and membrane (0.22), you will certainly get the band of your interested protein.

Best