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peptide expresion, but no western blot bands. - (Aug/21/2007 )

Hi, can you help me. I am running westerns of some small (30-40 aa) HA-tagged peptides. The peptides are expressed from a plasmid giving GFP expression from the same mRNA as my peptides (from an IRES). I have no problems seeing the GFP on a western of whole cell lysate, but when I do the western to see my peptide of interest I get a blank blot (my antibodies are working, and I know the peptide is made due to activity assays). I run the cell lysates in a 10% tricine gel and use the immobilon-psq membrane. I transfer in 20 or 30% methanol. Thus, I have done what I know of to ensure that the problem is not due to the small size - can I do something different? I am thinking, that maybe the peptides are degraded non-enzymatically (I have all kind of protease inhibitors in my lysis buffer, and the GFP is there). Can this be prevented? Any other suggestions what the problem may be?

Thanks,

-molbiola-

Because of the small size of the peptide, you should be careful about your conditions so you don't blow it through the membrane. If you setup your transfer so the large proteins are transferred efficiently, you'll blast your peptide out the other side. A quick check for this is to have a second membrane in place to catch anything that gets through the main membrane.

What kind of membrane are you transferring onto? Double-sided PVDF will work well for small peptides, and it holds them very well. Alternately, only transfer for a short time, or use a lower voltage

-swanny-

QUOTE (swanny @ Aug 22 2007, 01:36 AM)
Because of the small size of the peptide, you should be careful about your conditions so you don't blow it through the membrane. If you setup your transfer so the large proteins are transferred efficiently, you'll blast your peptide out the other side. A quick check for this is to have a second membrane in place to catch anything that gets through the main membrane.

What kind of membrane are you transferring onto? Double-sided PVDF will work well for small peptides, and it holds them very well. Alternately, only transfer for a short time, or use a lower voltage




Thanks for your reply. I am aware of the transfer issues with small peptides and thus use the Immobilon psq membrane. The cut-off of this membrane is lower than my peptides, so theoretically this should not be the problem. I can try the double membrane trick.

-molbiola-