Help with Western Blot!

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jxr's picture
Help with Western Blot!

Please help, I am new to western blot analysis.

I did a blot using beta-actin antibody and biotinylated protein ladder. I also used an antibody that worked for me before on the other side of the membrane (I cut it in half).

The beta-actin antibody gave no signal, and the ladder was very faint. I did use anti-biotin in my 2nd antibody. I know that my transfer and technique was good because of the other antibody I used, the ladder and protein of interest were correct.

Am I blocking too much? Could degradation of an antibody block signal of the protein ladder?

Thank you.