Molecular weight of my protein of interest is 7KDa. I am not able to get this on 18% SDS-PAGE electrophoresis. I want to do western blotting of my samples having this. Kindly suggest the method to separate the low molecular wt protein and western blotting of it.
Thank you
Theoretically, Tris Tricine buffer is used for separation of low molecular weight proteins (peptides).
Here is the link for info;
https://info.gbiosciences.com/blog/tris-tricine-sds-page-what-is-it-and-how-to-perfrom-it
Your peptide is probably running out of the gel. You may need to use a higher percentage of cross-linking, for instance 25%. I would prepare a gradient gel, e.g., 10 - 25%.
Also, how do you detect the band in the gel? What staining method do you use?
Sometimes certain peptides do not show up. For instance, proteins or peptides that are glycosylated may show up as negative, white bands in some standard staining methods.
by using higher concentrated gels. Maybe you can bye a pre-manufactured gradient gel or a higher concentrated SDS gel.
As Achim suggested, it is better to confirm whether your peptide is still in your gel after electrophoresis. You may use the loading buffer which contains BPB (bromphenol blue), and BPB runs prior to the low molecular weight proteins. Therefore, running the SDS-PAGE gel until the BPB dye is almost bottom end of the gel is the suitable running period from the point of avoiding the running out of peptide from the gel.
Next problem would be transfer step from gel to membrane. As you know, small protein can move faster than large protein, and sometimes these small proteins pass through the membrane.
Another suggestion. You are trying to do western blotting against peptide, but why do you need it? Do you want compare the amount peptide between the samples? If so, dot blot or direct ELISA can be used for comparizon. If you want to clarify the digestion of large protein into small peptide, you do need to separate them using gel.
As Hiroyuki mentioned, peptides move a lot faster than proteins. When I did transfers of peptides (long, long ago), I always stacked 2 or even 3 membranes. You may also try to try using different membranes, esp. try hydrophobic ones.
Achim Recktenwald Mol wt of protein is around 7KDa and length is around 51. so, is it still possible that it is going out of the gel?
Hiroyuki Kanzaki tricine gel will be better or AUT PAGE?
Further i want to proceed with western blotting. i think dot blots are generally used to study protein-protein interactions?
I suspect in a 10% gel, it is running with the bromphenol blue front or just behind it.
I would use a higher percentage cross-linked gel. If I am right, and it is within or very near the front, you will also have a lot of other small peptides running there, a lot of crap you do nbot want.
Tris Tricine buffer, as mentioned by Hiroyuki, would help, as well.
i think tris tricine gel is a better option to see the bands of low molecular weight proteins, i was also facing such problems the i tries tris tricine gel and it worked.
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