Dear colleagues,
I am working on a protein with peculiar characteristics. It is reasonably abundant in stem and cancer cells:
- Size: 410 kDa
- pI: 10.44
- Unusually rich in isoleucine and glutamine
And SDS-PAGE is quite challenging. I tried almost everything including
- 1% SDS (final) in Tris-Glycine transfer buffer, 30V, 16 hr, overnight, PVDF
- 4% SDS (final) in sample buffer
- 3-8% Tris-Acetate gel
I would appreciate any suggestions. (The name of the protein is ASPM.)
This sounds challenging indeed. But not sure what role high pI (and charge) per se has on SDS-PAGE performance. My guess is the conditions you are using may not be adequate to disrupt the tertiary structure to enable sufficient SDS layering on the protein. Are you not seeing any electrophoretic separation? Or is it not entering the gel at all (because of the limitation of MW)? If the protein is comprised of non-covalently bound subunits, you can increase the SDS% as well as the treatment temperature (for sample prep).
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