How do i measure the enzyme activity of human GBA in cos 7 cells that have been transfected with mutant plamids. After 24 hours of plating cells in a 12 well plate, I transfect cos 7 cells with pcDNA containg the GBA cDNA with the specific mutation. Transfection is done using Lipofectamine 2000 following the standard protocol with 2ug of plasmid DNA. After 48 -72 hours of transfection the cells are harvested and assayed for enzyme activity using the SIGMA GBA enzyme activity assay kit(MAK 129). How can i extract the proteins from the cells without denaturing it? I used 50mM phosphate buffer to extract the cells and then centrifuged. The supernatant was used for enzyme assay, but the activity was very low even in the wild type one..
please suggest how i can improve this assay.
Please give more details on the centrfuage speed. Why not using whole lysates using RIPA lysis buffer, I know it may affect on the enzyme activity. But I'm affried you lost most of your enzyme in sediment.
Try harvesting the cells 24-48 hours after transfection. Hope washed the cells three times 1X PBS or the buffer used before harvesting, check the pH. Also, use ice-cold buffer, and perform centrifugation at 4 degrees. Check the protein content in the supernatant.
@ Elsayed Metwllay Thank you for your answer. Centrifugation was done at 12,000rpm for 12 mins. i have also tried using RIPA lysis buffer but the enzyme activity was very low. So thinking RIPA buffer is too harsh i used th50mM phosphate buffer.
Please suggest as to how i can check the protein content in the supernatant
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