I am doing drug treatment in primary culture of astrocytes, in western blotting big proteins like GAPDH and Actin are prominently detected but smaller proteins like BDNF(17kda), GDNF(17kda) and S-100B(10kda) are not detecting. Steps which i am following are as follows:-
75 microgram samples
15% acrylamide gel
2 hour transfer (80v) and 1hr also tried
0.45 micron PVDF membrane
2 hr blocking with 5% milk
Now, what change should i made???
Small polypeptides can easily transfer through the membrane. I would use 0.2 micron membrane and use two sheets so you can perhaps capture the smaller proteins if they do go through. You can try decreasing the transfer time even more to keep the small proteins retained on the membrane. Use 10-20% methanol in the transfer buffer to keep the gel from swelling. And don't equilibrate the gel in transfer buffer (the proteins may diffuse out). Just rinse in buffer to wash off excess SDS, and make your sandwich. Some people have had success using formaldehyde to fix small polypeptides to the membrane after transfer
before proceeding with the blocking step.
There are a lot of hints on the internet. Try searching for "Western blot transfer of small proteins peptides" if you have not already done so.
I also do the western blotting for the 17kd protein, I got it. I used as following;
16% acrylamide gel,
Nitrocellulose membrane
Transfer 1h (100v),
1h blocking with 5% milk
You could try it.
Good luck!
Adding to the above answers, 12% gel, overnight wet transfer at 30V worked well for me for GDNF. Use TBS as working buffer for antigen-antibody probing. Not PBS.
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