Hello to everyone!
I want to see little proteins (15-20 kDa) by WB and for the transfer I have the semi-dry transfer blot Biorad. Generally, for a 10% gel I transfer at 80mA per gel for 1h and the transfer is good. Now, I had 2 gels at 15% and I did 160 mA for 1h. Unfortunately, after the transfer I still saw a good amount of marker on the gel and in fact the signal of all proteins was not good after the developing.
Do you have any suggestions how to solve the problem? Thank you very much!
Firstly, have you run an SDS PAGE coomassie stained gel of these proteins? Just to verify if your proteins are there and are at a decent concentration. Your proteins might require concentration before running on the gel.
Secondly, have you stained your nitrocellose/PVDF with Ponceau S before you block to verify protein transfer or do you rely on the transfer of the marker from the gel to nitrocellulose?
And finally, which model of the biorad blotter do you have? In our lab we have a sigma semi dry blotter and 20V for 1 h works like a charm. If you have a wet blotter from biorad, 20V per gel/nitrocellulose snadwhich for 4h works for my fussy, low concentration proteins.
Hope this helps
Since you are using pre-stained markers, you should have a pretty good idea if the proteins in your size range of interest are transferring, and usually the difficulty is more with the large and not the "little"! We have used the Life Technologies iBlot 2 system (which is dry, not semi-dry), not the Bio Rad, but one suggestion is to equilibrate the gel in 20% ethanol for 5–10 minutes prior to transfer; we take this step to help increase overall protein transfer efficiency.
Hi Carla
Transfer will never be 100% efficient and different proteins will transfer differently. If you do a coomassie stain after the transfer you will always see a lot of proteins in the gel no matter how you do the transfer. The best indicator of a good transfer is only the marker line.
Did you try to blot the membrane to see if your protein is there? Maybe you do not need to optimize further.
Moreover, which buffer are you using for transfer?
I stopped to use MetOH buffer because the transfer efficiency was really poor and I always see the 55KDa marker band still in the gel. I now changed to TBE buffer which performs better. I run 2 gels in the same machine at 320mA for 1:30 h, 25V max, and after the transfer the only marker band I see are higher than 200KDa. You can try to do blot in parallel using MetOH or TBE and see which one performs better for your protein. For 1 gel we use 160mA 1:30h for TBE and 100mA for 1:30 h for MetOH in the Biorad semi-transfer machine.
If you want to optimize your transfer for 15KDa proteins you should use a membrane with a pore size of 0.2µm in order to block protein diffusion. PVDF or nitrocellulose do not change the results. I prefer nitrocellulose because gives brighter results.
Hope these will help.
Best,
Edoardo.
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