I am trying to quantify a 6 kda protein using western blot. I am getting a band at 50kda. I am using a 15% gel and probing with a monoclonal antibody. Please help.
Very small fragments are often difficult to see on SDS-PAGE.
First, you can go to more than 15% for your resolving gel (up to 20%), and avoid to make the bromophenol blue get out of the gel to be sure that your band won't also get out.
Second, you can use Tris-Tricine gels rather than Tris-Glycine ones, this usually lowers diffusion for very small fragments.
Hope this will help.
Check your antibody data sheet, it shows the band pattern. Some times you find the non specific bands also. Of-course it is non specific band. You shd run gel until dye front reaches the 2 cm before end. It is very small weight so some times we lost with the dye front. This protein usually run almost with dye front. I did the same and got the results
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