I am preparing a protocol for microsome preparation from HEK (human epidermal keratinocytes) and would like to know if anybody have experience in this kind of procedure.
Like CYP concentration is known to be very low in this type of cells, I think a very high amount of cells (maybe 200.10^6 cells) are needed to prepare a single sample.
It would be great and very helpful if somebody could share his experience.
Thank you
Paul
Dear Paul. You could prepare easily 20-25 mg of microsomal protein from properly homogenized 1.0 gr (wet weight) rat liver. To detect and quantify P450 amount (in nmole/mg microsomal protein) you should take 0.4-0.5 mg of microsomal protein per ml of media to have nice CO spectra. If P450 amount in microsomes of keratinocytes is for example, 20 fold less than that in the liver (which is most likely), so ... Additionally, you have to keep in mind that keratinocytes might not to be rich in endoplasmic reticulum (biochemically, microsomes) as liver cells. This is really very difficult task to detect and quantify P450 according CO-dithionite spectra in various organs and cells except, liver, kidney, lung and, may be, spleen. Good luck in your efforts. If you need do not hesitate ask me questions.
Sincerely
Hello Vladimir
thank you very much for your advises.
For the time being, I have measured CYP enzymatic activity data on native keratinocytes, activities which are also highly inducible by 3 methylcholanthrene, so for a characterization purpose I need to isolate the CYP.
I have planified to try the method you've mentionned to quantify CYP with CO spectra.
Why do you say that this method is difficult in other cells than those you've cited? Only beceause of low expeted levels of CYP? (that's why I want to use a very hight amount of keratinocytes to strongly concentrate the CYPs)
Best regards
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