Hi! I did a lot of western blots and I always had high-intensity background on my membrane.
So, before I did western blot, I did SDS-page electrophoresis. The gel consisted of 10% running gel and 4% stacking gel. I put sample buffer (SDS+ beta-mercaptoethanol) until the end concentration was 1x SDS+BME.
The membrane I use was PVDF. After transfer, I blocked the membrane with PBST: Odyssey buffer (1:1) for 1 hour and incubated the membrane using primary antibody diluted in PBST-Odyssey buffer (1:500) overnight. At the next day, I washed the membrane 3x using PBST, 5 minutes each. Then, I incubated with secondary antibody diluted in PBST-Odyssey buffer (1:5000) for 1 hour. I washed the membrane 3x with PBST, 5 minutes each before have it scanned.
I succeed with this protocol for 3 times. And after that, every time I did western blot, it always end up with high-intensity background in the upper part of the membrane. And there was a line at 55-70 kDa.
Is there something wrong with the protocol? I suspect that there was something wrong with sample buffer. But, I made the new one and the results were still the same.
I blocked the membrane with PBST and Odyssey buffer (1:1) for 1 hour
Did anything else change from the three times you succeeded to now?
I have always just used 5% dry milk in PBST as a blocking reagent and 1% milk in PBST to dilute my antibodies. If you are set on using the Odyssey buffer (which I have never used before so I cannot speak to its efficacy or its shelf life), try diluting your antibodies more.
I also recommend blocking overnight and incubating with primary antibody for 1-2 hrs, depending on how specific and strong the antibody is.
Also, what are the antibodies you are using?
Hi Hegar,
I am also using the LI-COR Odyssey system. The manual suggests never to use Tween before or during blocking. I highly recommend to dilute your Odyssey Blocking Buffer in PBS only. In addition, are you using PVDF-FL with low autofluorescence?
Regarding your problem: I would check whether my antibodies cross-react or are contaminated. Scan your membrane directly after transfer to rule out any problems arising from the protocol until to this point. Then incubate with primary or secondary only and scan again.
Hope I could help a tiny bit.
Thomas
Dear Hegar,
I think that your instincts are correct, and there is something odd about how the samples are running in the gel. Perhaps from the sample buffer, the preparation of the samples themselves, or even a problem with the gels. To get a handle on this, have you tried to stain the gel itself (don't bother to do a transfer) with Coomassie Blue or something similar? If you see a similar smear in the gel, then it is not a problem with your antibodies.
I am not familiar with the Odyssey system., so I am not sure what Thomas means when he suggests you scan the membrane directly after transfer. However, some investigators pre-stain their membranes with SYPRO Ruby to asses transfer before trying to detect their protein of interest. It might be a step you consider adding until you get the problem solved.
Good luck!
Jill
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