I am trying to detect Sec61-gamma with protein size 7.5 kDa. Does anybody have suggestions for me on working with such small protein size? What advice you give me regarding the process?
First, make sure the protein band is well-resolved on SDS-PAGE. You will need to use a high-percentage gel, or a gradient gel, or a Tris-Tricine gel.
You may have to shorten the transfer time from what you would use for larger proteins.
There is a risk of the protein passing through the pores in the membrane. Use a 0.2 µm pore size instead of a 0.45 µm pore size membrane. That might reduce the effect somewhat, although the pores are still much larger than the protein.
Consider trying a semi-dry transfer instead of a wet transfer system.
This protein also has a theoretical pI of about 10. You may want to take that into account as well.
We didn't have much success with IB of such small proteins with Mr around 10kDa. Can you explain a bit the purpose or following step after the immunobloting? If it's only for semiquant, then there are other ways to do it.
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