Hello,
I have been having issues with my western blot SDS protein extraction buffer and contamination. I followed the recipie from Thermofisher with 2.5mL 0.5M Tris HCl (pH 6.8), 2mL glycerol, 4mL 10% SDS. When working reagent is added, it turns purple rapidly. It seems that decreasing the glycerol by 50% decreases the concentration of contamination, so I am leaning towards the issue being with our glycerol stock. Does anyone know/ have advice for determing the soruce of contamination, or a better SDS buffer recipie to use?
Thank you!
Hello,
It's possible that the issue could be related to your glycerol stock, or there could be contamination in one of the other reagents. Here are a few suggestions for troubleshooting the problem and potentially finding a better recipe for your SDS buffer:
- 2 mL 0.5 M Tris HCl (pH 6.8)
- 1.6 mL glycerol
- 2 mL 10% SDS
- 0.5 mL 0.1% bromophenol blue
- 0.4 mL 2-mercaptoethanol or dithiothreitol (DTT)
Remember to adjust the volumes according to your desired final volume.
I hope these suggestions help you resolve the issues with your SDS protein extraction buffer. Good luck!
Thank you for your suggestions! Very helpful answer. We are working through it now. Also, the working reagent is 50 parts + 1 part thermofisher pierce protein assay reagent A:B.
You may have exceeded the compatibility limits of the BCA reagent with the components of the buffer. Here is a compatibility chart:
https://tools.thermofisher.com/content/sfs/brochures/TR0068-Protein-assay-compatibility.pdf
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