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invivoVibrio 03-29-2012 06:24 PM

Western blot mystery - possibly a sample preparation problem?

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 a protocol I found, briefly: cells are lysed in 2% 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!


neus3z 04-26-2012 03:32 PM

Re: Western blot mystery - possibly a sample preparation problem?
Hi Mark,

I've done Bcl-2 a lot of times in my PhD, and I always used 15% SDS-PAGE gels. I've never used 2% SDS but if you put the appropiate amount of protease inhibitor it should work fine. You can also try with RIPA, by using 20 ul RIPA 1x / million cells. Of course, all the process at 4, with ice. After incubating for 20 min I centrifuge the samples at 4, 10000 rpm 10 min and save the supernatant (quantification at this point, before adding the load buffer with bromophenol and glycerol). I boil the samples at 100 5 min, a brief spin and ready to load into the gel.

I'd try to run the gels at 40mA/gel (or 120-140 V constant). In BioRad system it should take about 90 min with a 15% gel. Be careful with the running buffer and check the recipe. Of course, also with the transfer buffer, if you are using PVDF membranes you need to activate them with Methanol (absolut ethanol works fine too), and the transfer buffer needs 20% methanol if you use semidry system from BioRad. If you use wet transfer it's not necessary to add it to the buffer you are going to put into the tank.

I hope you are getting better results...

jeeep 06-23-2012 11:20 PM

Re: Western blot mystery - possibly a sample preparation problem?
Are your precast 4-20% gels Tris-Glycine or Tris-HEPES? The manufacturer will have a recipe of the lysis buffer, just add the protease and phosphotase inhibitors and proceed with how neus3z recommends. I've found that only the lysis buffer recommended by Bio-Rad will work for my 4-20% Tris-Glycine gels.

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