Hi,

I'm running an immunoprecipitation experiment, trying to pulldown actin from some RAW 264.7 cell homogenates.

I ran a Western blot with a 1:5000 dilution of biotinylated anti-actin antibodies and reacted it with AP-SA (1:5000 dilution) and BluePhos solution.

The WB detected actin in the crude cell homogenate and wash, but the pure actin control lane was blank, despite a clear band appearing in the silver stained gel.

What are some the possible reasons for the pure protein control not appearing in the western blot. Previous WBs detected the pure actin just fine.

Should I reprobe the same membrane with HRP-SA and ECL solution? I heard that's more sensitive, but would the antibodies from the previous WB interfere with the ECL detection. I have tried to strip the membrane with a low pH buffer but to no avail.

Thanks in advance for the help.