I am currently working on beta-endorphin (3.5 kDa). I made a 10% tricine gel with the modified method (Haider 2012) which works quite well for my molecular weight marker (GE Healthcare) and the recombinant beta-endorphin (in 0.1 % acetic acid). But my samples extracted in RIPA buffer or different tris based-buffer migrate only until 12 kDa.
Can you offer advice on a more appropriate extraction buffer for separation in a tricine gel ?
Hi, Please check the link, It may be useful for you.
http://depts.washington.edu/bakerpg/protocols/tris_tricine_gels.html
Thanks for your answer, in fact I already know this protocol and my problem is the protein preparation that is not explain in this protocol.
Has your method worked before or did you just start this experiment? Perhaps you protein band is not your target band or if it is, it is that size due to various protein modifications? Perhaps try a blocking peptide to see whether your band is your target protein or not. I doubt the problem is with the gel. look at your extraction method to ensure you get the right protein but if you based it on published protocols than that should be OK. Although the size difference is fairly large between 3.5 and 12KDa, variations in your sample/cell type can sometimes account for size differences in what is given by the antibody supplier and what you get.
Try with a 16% tricine gel. Look at this page:
http://www.komabiotech.com/product/product_detail.php?item=KG6045
Another thought. Are you sure that your small band did not migrate off the gel?
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