3 replies to this topic

[nelssara]

Hi! I was wondering if any of you could help.

I attempted to run a dot blot last week to optimize antibody concentrations and dilutions for a new developing reagent. However, after developing the blots there was nothing (NOTHING!) but background.

I dotted 1-5 uL of a protein lysate (so about 1-5 ug of protein) onto damp PVDF membrane (wetted in methanol for 15 s, MQ H2O for 2 minutes, and PBS-T for 5 minutes) and then allowed the membrane to dry fully. When I rewet the PVDF in methanol I could initially see the dots of lysate on the membrane, but then they disappeared. I'm worried that the protein did not fully penetrate the PVDF and was subsequently washed off during the rewetting step.

My lab does not have a dot blotter, so I'm stuck with manual methods. Any pointers or suggestions would be greatly appreciated.

Thanks!

[bob1]

PVDF should bind the protein quite strongly, so I wouldn't be worried about washing the protein off.

Can you tell us the proceedure that you used to probe the blot? - to me it sounds like you didn't block the membrane properly before the primary antibody step.

[mdfenko]

did you treat the protein as you would for a western blot (ie sds sample buffer to denature)?

if not, is your primary antibody good for native as well as denatured protein?

[knuf]

whenever i do dot blots i use nitrocellulose membrane...then you don't have to worry about wet/dry issues