The proteins I have studied so far have been confirmed by electrophoresis without any problems and all have positive positive charges. And also, markers sold on the market also consist of proteins with positive net charge. However, the peptide I am currently studying for have a net charge of -2 and a theoretical PI of 3.5.This peptide has problems that have not been confirmed by the electrophoresis method that has been performed so far. Is there a way to identify a peptide with a net negative charge by electrophoresis?
What kind of gel electrophoresis are you working with? On SDS-PAGE the net-charge is covered up by SDS so it should not be a problem (I have worked with a small 10 kDa Protein with a net-charge of -18 which I could readily identify). However, this may depend on the length of your peptide.
A comprehensive protocol for Tricine-SDS-PAGE of small proteins and peptides down to 1 kDa can be found here: https://www.nature.com/articles/nprot.2006.4
For shorter peptides HPLC would be the method of choice if it's available in your lab (since HPLC is usually run under acidic conditions a low pI is preferrable to get good retention on a reversed phase column).
If you have to do native gel electrophoresis (w/o SDS), the standard method has the anode (positive) at the bottom of the gel so you should ONLY see acidic proteins. Assuming you are running it the opposite direction - with the cathode (negative) at the bottom - you could just reverse the voltage (i.e. switch the red and black electrodes at the power supply) to see negatively charged peptides/proteins.
Kind regards,
Lorin
Dear @Lorin Steinhäuser
Thank you for your reply.
In fact, to date, I have identified a 1 kDa marker peptide(Bradykinin, 9mer) using 16.5% tris tricine SDS gel.
However, target peptides similar to this molecular weight have not been identified.
So I want to try the experiment methods that you kindly recommend.
1. What percentage of gels did you use for SDS PAGE?
2. If I try native gel electrophoresis, what should I do with my EzCell Vertical Electrophoresis System?
In this system, the anode and the cathode are located in the same direction, and if they are connected in the opposite direction, the sample will escape in the well direction.
Anyways thank you so much for your time.
I really appreciate it.
Regards
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