I wish to see different levels of these two proteins with normal SDS-PAGE (and not with other protein separation techniques). All post-translational modifications must be preserved. The proteins are highly soluble but do not tolerate acidic media. The samples are pure on these proteins.
The tris-tricine buffer system is significantly better than tris-glycine or MES with tiny proteins. This method uses separate cathode and anode buffers.
You'll really want to elevate the concentration of the acrylamide as well as the concentration of the crosslinker bisacrylamide. Introduction of something like 4-6M Urea in the composition can also help significantly with resolution of similar-MW proteins. Simple high-acrylamide gels won't benefit you because they lack the stacking resolution.
I have been using the attached protocol for a few years and have been really happy with resolution of 3-8kDA peptide fragments. It should be a good place to start!
Good luck.
If you are just trying to visualize these proteins on a SDS-PAGE gel I would suggest increasing the percentage of acrylamide you use in the gel. The higher the percentage the greater separation you can get for a lower MW. We use 16.5% gels instead of 12% gels to resolve proteins around 8 kDa. I realize that is much larger than 3.4 kDa, but the same principle applies. As far as staining or visualization techniques are concerned, that is going to be dependent the concentration of protein and your personal preference.
Higher percentage gel for lower molecular weight products as mentioned. You will also want to be careful not to run the products at ~3kDa off of the gel. Hence, you will need to stop the gel well before the dye front reaches the bottom of the gel.
The tris-tricine buffer system is significantly better than tris-glycine or MES with tiny proteins. This method uses separate cathode and anode buffers.
You'll really want to elevate the concentration of the acrylamide as well as the concentration of the crosslinker bisacrylamide. Introduction of something like 4-6M Urea in the composition can also help significantly with resolution of similar-MW proteins. Simple high-acrylamide gels won't benefit you because they lack the stacking resolution.
I have been using the attached protocol for a few years and have been really happy with resolution of 3-8kDA peptide fragments. It should be a good place to start!
Good luck.
I think with "normal" SDS PAGE (you mean a tris/glicine?) you would really have a hard time to see such a smal difference. 18% gels might also not really help you.
I read about tricine gels for separation of
Many years ago, I used the following protocol for peptides less than 10kDa.
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
Schägger & von Jagow Anal Biochem. 1987 166 368-79.
PMID 2449095
The tricine gels do give you better separation (take a look at the Shagger paper above) but I doubt you will be able to separate a 3.2 kDa from a 3.4 kDa. One issue is that the size/mobility relationship is no longer strictly linear for short peptides. Staining of short peptides is also an issue (frequently not linear with concentration). Would a 2D isoelectric focusing-PAGE gel work?
Indeed only 2D-gel can separate such as small different proteins, for sure they have different pI values. Good luck !
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