I am a student currently working on a summer project involving MIN6 cells. Part of my project involves measuring ER stress, which means I have to do many Western Blots. However, I am coming to the end of my time available on the project and I am still no closer to solving an issue with excessive bands appearing whenever I scan the blots, so I would greatly appreciate any suggestions. (Ive attached an actin blot I got several weeks back).
Over the weeks I have obviously tried changing a few things. We changed blocking agents (Milk and BSA), antibody concentrations (the actin blot was with 1:5000 primary and 1:10,000 secondary), all the buffers (running, transfer and TBST), lab temperature (which was around 10 degrees for a number of weeks) and protease inhibitor concentrations that we add to the lysis buffer (Benzamadine and PMSF). None of this has solved the issue.
My supervisor believes some of the following issues may be the cause:
*Our benzamadine stock that we add to the lysis buffer has precipitated out a bit from solution, suggesting it still may be a protease issue
*The Laemmeli buffer that we use for lysates may not contain enough SDS (I am making some more fresh buffer in a few minutes)
*Our protein samples weren't heated enough-we heat at 70 degrees for a few mins on a hotplate. Is it worth increasing temperature/duration?
Many thanks for any help anyone can give.
1) I would suggest to use fresh-prepared benzamidine solution every time. Please see the enclosed link:
http://www.sigmaaldrich.com/catalog/product/sial/b6506?lang=en®ion=US
2) It would be better to use commercial protease inhibitor cocktail (it contains inhibitors against all main groups of proteases, and you don't know which proteases are present in your extracts).
3) Benzamidine is a reversible inhibitor of trypsin, trypsin-like enzymes, and serine proteases and this is only one group of proteases. PMSF is irreversible inhibitor of the same group of proteases.
4) I would recommend to boil your sample in the SDS-Sample buffer at 100°C for 3 min before loading on the gel.
1) I would suggest to use fresh-prepared benzamidine solution every time. Please see the enclosed link:
http://www.sigmaaldrich.com/catalog/product/sial/b6506?lang=en®ion=US
2) It would be better to use commercial protease inhibitor cocktail (it contains inhibitors against all main groups of proteases, and you don't know which proteases are present in your extracts).
3) Benzamidine is a reversible inhibitor of trypsin, trypsin-like enzymes, and serine proteases and this is only one group of proteases. PMSF is irreversible inhibitor of the same group of proteases.
4) I would recommend to boil your sample in the SDS-Sample buffer at 100°C for 3 min before loading on the gel.
I completely agree with Viktor. I used to boil my samples at 95 degrees for 10 minutes in loading buffer containing Beta-mercaptoethanol
James: On top of the suggestions made by Victor and Andrea, I'd suggest to conjugate your primary antibody to HRP and to avoid using secondary HRP-IgG. We've obtained single band for all plasma proteins lysed from hepatocytes and other cultured cells with buffer containing no protease or only AEBMSF. Check out our paper at PMID: 25900303 or download from my RG.
Thank you Victor and Andrea for your insight. I will use a higher temperature for the sample heating before loading into gels (i'm assuming). For the protease inhibitors, our lysis buffer contains EDTA and EGTA for metalloproteases, but i believe previous researchers investigating ER stress proteins havent found the need to add any other protease inhibitors
Thank you also Ke-Wei. I believe we mainly use the indirect method to amplify the signal, however I will look into using only Primary antibodies.
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