Dear All,
I am trying to detect synthesised peptides with SDS-PAGE. The peptides migrate with a big difference (larger) in SDS-PAGE gel in comparison with their real molecular weight. And they refused to migrate in a single band, which makes things even worse. Do any of you have suggestions for improving the detection, please?
Thank you.
Dear Lauisa,
My peptides are ranged from 2 to 4kd. There is no Cyc in the sequence and I used DTT heating step. I do not understand what you mean to deprotect peptides.
They migrate to 6 to 20 kd with normal SDS-PAGE. I am thinking about to try Tricine-SDS PAGE. And may also lower SDS concentration in Running buffer, run in a lower voltage.
It will be helpful if you share your expertise.
Thank you.
ziguo
Before I can answer your question in detail, could you tell me the range in molecular weight and whether or not cysteine is pressent in your seuqence.
Secondly, have, you completely deprotected your peptides?
Thirdly, have you done a MALDI- TOF mass spectrum?
Dear Lauisa,
My peptides are ranged from 2 to 4kd. There is no Cyc in the sequence and I used DTT heating step. I do not understand what you mean to deprotect peptides.
They migrate to 6 to 20 kd with normal SDS-PAGE. I am thinking about to try Tricine-SDS PAGE. And may also lower SDS concentration in Running buffer, run in a lower voltage.
It will be helpful if you share your expertise.
Thank you.
ziguo
Maybe I misunderstood, your question did you mention that you have synthetic peptides (chemically synthesized)? If so, sometimes they do not get completely deprotected, and some problems may occur during SDS-PAGE. For peptides around 6,000-10,000 KDa I have used 15 to 20% gradient gels in a Tricine buffer. If you check the BioRad catalog on-line they present many helpful tips and figures to see what to expect. Keep the same recommended SDS concencentration. Perhaps the 10-20 Kda peptides can be separated on a 12-15% gradient gel. Gradient gels are the way to go, along with the appropriate standards.
Thanks Louisa,
My peptides are chemical synthesized.
Is SDS micelle a problem for SDS-PAGE? Could urea in the system help for a better migration? I feel I am luck of expertise in this field.
ziguo
I highly recommend that you perform a MALDI-TOF on the peptide mixture to make sure the peptides are of the expected molecular mass. Urea that is freshly made up will help, but do NOT use old urea solutions as they will cause carbamylation of your peptides, thereby changing molecular weight, function and electrophoretic mobility. It is possible the peptides are aggregating as well.
I think that you can go on with the tricine-sds gels. I performed the gel as stated by the original paper from Schägger (http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.4.html) and it worked well for me with peptides within the same range as yours. Also, including fresh urea might increase the resolution of the gel.
Thank you, Alessandro Trentini. tricine-sds, fresh urea addition, and run really slow helped.
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