Western blot mystery - possibly a sample preparation problem?

Hello!

I've been trying for a few weeks to run some western blots in my lab, with limited success.  I really need to get it working (my PI is getting very annoyed with me), but I'm having a hell of a time figuring out why it isn't working.  I'm the only one in my current lab running westerns, so I'd really appreciate some help!

The samples I'm using are from total cell lysates of rat spleens, mouse thymus, and MCF10A human cells.  I've been able to detect actin and tubulin, though the detection isn't as high as I would like.  That may be due to an old antibody, though.  The bigger problem is that I haven't been able to detect Bcl-2 in my samples.  It's a smaller protein (~25kDa), which may be part of the problem.  Using a positive control mouse spleen sample from Santa Cruz Biotech (where my antibody is from), I've gotten a Bcl-2 signal, so that suggests it has something to do with how my samples are prepared.

I'm preparing my samples according to this: (http://www.biobanks.se/documents/Protein%20Extraction%2020030807.pdf), briefly: cells are lysed in SDS (basically laemmli buffer without bromophenol) by pipetting and vigorous vortexing, then incubating at 70 degrees for 20 minutes.  Quantification of my samples by BioRad RC/DC puts them at about 2mg/mL.  I've been loading 20ug per lane (15uL well sizes, but I don't like overfilling them).

An observation that may be related is that the blue line in the running gel runs very quickly through to the bottom, and gets very dispersed.  The bottom few markers on my protein ladder are also smearing.  I've tried using gels from 8% to 12%, and varying the voltage from 125 to 200, and nothing seems to have an effect.  I've ordered some pre-cast 4-20% gels to see if that helps.

Does anyone have any ideas what may be going wrong?  I've been frustrated with this for far too long, this *should* be a real simple, quick assay and I should have been done with it weeks ago.  I think my PI is going to be very annoyed if I don't have any data for him before he leaves for AACR this weekend!

TIA

Mark

Hi Mark,

I have not used the method you mentioned to prepare sample for WB, so it is pretty hard to tell how it might go wrong. However, I routinely use RIPA buffer to lyse cells and an electronic homoginizer to aid protein extraction from tissue samples as depicted  in this link (from Abcam). Also, if you have not done so, adding protease inhibitor cocktail in your lysis buffer may help too.

Also, based on your observation of the smearing in your gel- Did you pour your own gel? Did you get the same smearing pattern when you run the control from Santa Cruz? And what is the concentration of this control? And how much did you load for you to detect the protein? Is the amount much higher/lower than your real sample?

Cheers.

Thanks for the reply!

I've used a protease inhibitor cocktail in one extraction, and I didn't see any difference in bands after coomassie staining, and the western didn't show any band.  So I don't think that's the problem.  70 degrees in 2%SDS *ought* to kill any proteases.

I've been pouring my own gels.  The control sample is 2.5mg/mL, so I've been loading 25ug.  The dye front on that sample was hard to see, since they used a smaller amount of bromophenol, but it appeared to smear just as much as my other samples.  From RC/DC quantitation, the control lysate is almost equal concentration with my samples.