I ran a Western earlier in the week and when I read my result, there were ghost bands, non-specific bands, and a dark background. At first we thought I loaded too much protein (40 ug on a 0.45 um), but when I ran the control (beta actin) to check, the bands weren't blown out. The only thing I could think of is that I readjusted the membrane slightly before the transfer and used thinner sponges for the first time.
I used 1:1000 primary and 1:5000 secondary, for two and one hour at RT, respectively. I wash for 5 min three times and use ECL. I read somewhere that if your gel box isn't aligned evenly I could have issues. Any help would be greatly appreciated.
Hi Regan,
issues like the ones you observed can also be caused by antibody degradation. Did you use a fresh aliquot or an aliquot that was already used (and probably frozen/thawed multiple times)? While it is possible to re-use antibody aliquots this can also lead to problems like blot membrane darkening, unspecific binding, etc.
When re-using antibody aliquots it is important to store them cooled (e. g., at 4 °C) but not frozen.
I hope this helps you a bit.
Regards, Christian
Hi Christian,
I made a new aliquot for this specific blot, however I'll have to double check the expiration of the original antibody. I keep the antibodies in the freezer, then after I make the dilutions in 10% milk I keep them in the fridge. Thank you for your advice, I really appreciate it!
Hi Regan, looking at this blot, I would say that all of the staining is non-specific.
The MW standards are showing up clearly and distinctly in your blot so it is not a readjustment problem.
Because of the standards showing up so strong, I would say that you need better blocking reagents (maybe 10% BSA or 10% serum) and/or better primary/secondary antibodies.
Hi Steingrimur,
I use 10% non-fat dry milk for one hour at RT, so do you think BSA could be a better alternative? I use Cell Signaling Technology antibodies, which I've heard are of good quality. I've been able to detect this band with this cell type before, so could the issue be with the protein lysate do you think?
Thank you for your help!
Hi Regan, everybody has their own preference for blocking buffers. Personally, I like BSA because it is a "sticky" protein and it is is cheap.
There are a host of issues that might be interfering with your WB. Like Christian said, it might be an issue with the Ab.
But take a look at your previous WB. I wouldn't trust blots where MW markers show up.
This is what the gel looked like during SDS-PAGE. I've never had such wide bands and the one side is higher than the other. I've read that this means too much protein is loaded, but would then why wouldn't my control bands be blown out?
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