Bands Not visible on Western Blot

Protein Detection Western Blotting
1

Hello,

I have been trying to do a western blot. The problem is that my molecular markers show up but none of the bands do. However, think that some bands are very slightly visible - you have to look at it very closely. Also, my blot is very patchy. I incubated my membrane in 5 %milk solution for 1 hour. Incubation in primary antibody was for 2 hours at room temperature. This was followed by incubation in secondary antibody at room temperature for 1 hour. Please let me know whatever ideas you have that can help me resolve this problem.

Thank you.

2

Nishan

In your early days with Western Blotting, Its usually a good practice to run your sample in duplicates and then stain one half of the sample with CBB G250 or R250 and use the other half for transfer on membranes and use for your blotting. If samples arent even visible on the gel, you know the source of error

3

Thanks for your advice, buddy!