Dear all,
I'm facing a protein-binding issue in my final product. I currently add BSA to protect the protein and avoid the binding to the polypropylene tube. However, I can't do any UV280 analysis of my product with BSA addition, and I need to replace it by something else. I tried to remove it and just use a low binding polypropylene tube for storage at -80 degrees, but that's not enough and I still lose a lot of my product, which is already very diluted.
I heard about 5% Threalose or 0.05% Tween20, or siliconizing polypropylene tubes. Any other suggestions of conditions I could try?
Thanks a lot for your help!
Gelatin has been used for this purpose. An advantage of high-purity gelatin is that it does not have a UV chromophore, so it does not interfere with UV measurements of the other protein.
You may use the blank subtraction approach by preparing a reagent blank that has a composition of BSA at a concentration that you used to prevent binding and other medium components. After measuring the reagent blank absorbance you can subtract the obtained value from your sample absorbance to get an accurate (relatively) response of your sample at UV 280nm.
If it is not applicable for your assay anyway, then you may change the carrier agent with tritonx100, tween, or gelatin as the authors suggested. It is crucial to select the low binding tubes properly, especially from well-known brands.
Thank you very much for your answers!
Adam B Shapiro which amount of gelatin would you recommend to try?
Sebastian Schmitt
unfortunately we store our product at -80 degrees, and the glass vials usually can't go further than -20 degrees without being damaged...İsmail Emir Akyildiz that's an idea we have previously tried but our product is so diluted that the BSA covers everything... We are also using specific low-binding polypropylene tubes for the storage.
Thanks for sharing your ideas!
Copeland mentions having success with 1 mg/ml gelatin in his book Enzymes, a Practical Introduction to Structure, Mechanism and Data Analysis, so I suggest you start there.
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