I am currently working on developing a fast/cheap assay of a certain protein in the blood that happens to be an excellent biomarker for a variety of diseases. I plan to be able to run my assay using a single drop of blood. I am running into issues because catalase is interfering with the reaction, and I need a quick and cheap way to separate catalase from this protein of interest. Right now I am considering "salting out" the catalase with ammonium sulfate and running my assay using the resulting filtrate, however, if even a small amount of catalase leaks through, it will disrupt the assay. Can the total elimination of catalase from the sample be accomplished with the simple addition of the correct molarity of ammonium sulfate and filtration using filter paper? Or are more advanced filtration techniques needed?
I am also open to other suggestions for how to neutralize/remove catalase, I have tried numerous chemical inhibitors/denaturants (most interfere with the reaction), size exclusion chromatography (too expensive given how similar in size these proteins are), and changing the pH of the assay solution (both proteins work optimally at similar pH values).
Thank you for your time!
William Raskopf
Hope this will help
Article A Simple Outline of Methods for Protein Isolation and Purification
Dear doctor I think that the article of my colleague Kashif Ameer is very valuable and good indicator for your running way. And have a good luck.
Serum Protein Electrophoresis (SPEP):
https://www.gbiosciences.com/image/pdfs/protocol/BE-406_protocol.pdf
http://www.bu.edu/picf/files/2010/10/hofferelectrobook1.pdf
Can you immobilise the catalase with an antibody possibly bound to an inert matrix?
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