I ordered a synthetic peptide (sequence = MHHHHHH)
This peptide has a molecular weight of 0.97 kDa based on a web-based prediction tool and mass spectrometry.
However, when I ran it on the SDS-PAGE, I can see the protein bands around 10~15 kDA
What can be potential problems?
The picture shows the result of my 16.5% SDS-PAGE.
I ran the SDS-PAGE a little bit because the peptide was too small and I thought it might escape from the SDS-PAGE
First of all, you wasted ~ 80% of your gel, you could run it much longer. Only then it would make sense to look at the peptide band.
If you have mass spec data for the exact mass, then it makes no sense to run a gel unless you want to analyse exactly that what you observe: the anomalous behaviour of a peptide with mainly histidines which is not surprising that it behaves differently compared to a "normal" peptide with multiple different amino acids. If you just want to know the mass, go for mass spec.
The concentrating effect of discontinuous electrophoresis ensures that even proteins at the bottom of the gel run in tight bands. You can further improve that by running gradient gels. In addition, for small proteins, I'd use tricine electrophoresis (doi:10.1038/nprot.2006.4) for better resolution. With all these improvements, you should be able to get a better estimate of the size of your protein. However, note that electrophoresis is not a first principle method for molecular mass determination, not all proteins are on a straight line on a Ferguson-plot.
First of all, you wasted ~ 80% of your gel, you could run it much longer. Only then it would make sense to look at the peptide band.
If you have mass spec data for the exact mass, then it makes no sense to run a gel unless you want to analyse exactly that what you observe: the anomalous behaviour of a peptide with mainly histidines which is not surprising that it behaves differently compared to a "normal" peptide with multiple different amino acids. If you just want to know the mass, go for mass spec.
SDS-PAGE can only resolve proteins larger than SDS micelles (the front in your gel). Additionally your small peptide might be very soluble and partially escape your staining. For such small peptides MS is the best analysis method. It's quick and doesn't need much sample. Ask someone to run a MALDI or ESI (LC/MS) for you...
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