Western Blot TroubleShooting

[mutant11]

Dear all,

I need some help with Western Blots. I am trying to detect a transcription factor that is expressed at pretty low levels endogenously in cell lines. Does anyone have any suggestions?

-I use RIPA buffer with protease inhibitor to harvest the protein. I keep everything on ice.
-I incubate in primary antibody + block overnight.

Thanks!

[daniel.kurz]

You need to give more information on your procedure. What antibody are you using, how are you separating (native vs. SDS-page gel), what kind of transfer are you doing, etc etc.

[mutant11]

I harvest my samples using RIPA buffer + protease inhibitor on ice. I use a 6 well dish and I scrape the samples off on ice. I rotate the samples at 4C for 1 hour for lysis. I then centrifuge for 30 min. at 13,000 rpm at 4C. I take off the supernatant. I add 2X SDS PAGE loading dye and heat at 100C in a sand bath for 10 min.

I run my samples on a SDS-PAGE gel (7.5%) for 45- 1 hour at 150 V. I use the SDS-PAGE ladder as a guideline for how long I run the gel. I transfer onto a nitrocellulose membrane. I use a solution that is transblot solution, methanol+ distilled water. I let my gel sit in the transblot solution for 15 min before I start the transfer. The transfer is done in the cold. I block in 5% milk + TBST. I have tried two polyclonal rabbit primary antibodies against my protein of interest. I use an antirabbit-HRP secondary antibody.

[daniel.kurz]

That all sounds quite good. What is the problem that you are facing? What is the dilution of your anti-bodies? Your time for centrifugation may be a tad bit long since you're only looking to get rid of large intra-cellular junk as a result of lysis.

[mutant11]

I am not able to detect endogenous levels of the transcription factor. I was wondering if you had any suggestions on improving the signal of my protein. Would it be worth it to do a nuclear lysate?

[daniel.kurz]

If the transcription factor is below the levels of normal detection (which is quite frequent), it won't show up on the gel unless you really jack up the antibody levels at the risk of more background. Here's what I suspect is happening: the transcription factor is getting caught in the nuclear membrane parts and getting dragged down in your spin. I would go back and think about doing a nuclear prep using Current Protocols as a guide. Millipore also has some good information on the subject. I'm thinking that you're not getting your transcription factor solubilized away from the "junk".

[mutant11]

Thanks for the advice Daniel!

[sekibio]

If the concentration of your target protein is too low, you could consider using immunoprecipitation.