I express recombinant protein in yeast. After that harvest cell and resuspend cell in Phosphate Buffer Saline (PBS) buffer with 1 mM PMSF (as protease inhibitor), then break it by glass bead and bead beater machine.
Then, I dilute protein with PBS buffer, and coat it into ELISA plate.
(Indirect ELISA method). And I always coat it for over night at 4 deg celsius before use.
The Question are
1. Should I add 1 mM PMSF (final concentration) into dilution buffer (PBS buffer) and use it directly to coat the plate or not? for protection of protein degradation.
P.S. This PBS buffer use for dilute protein before coating well, and also use as negative control
2, PMSF in dilution and coating buffer interfere indirect ELISA or not? (For example interfere for protein binding to plate)
Thank you
Adding PMSF is unlikely to affects your ELISA. However, it has relatively short half-life depending upon the buffer and its pH.
As Divaker said PMSF has a short half life of approximately of 35min in aqueous buffer solutions at RT.
If you suspect possible degradation of your protein by serine proteases, you may try adding serine protease inhibitors like pefabloc which are more stable in buffers than PMSF. Out of curiosity, have you checked if your proteins are degraded during overnight incubation?
Dear Choubey ,
Thank you.
Dear Nandana,
I will check for degradation of protein. I also don't want to add PMSF into dilution buffer because the smell is very strong.
Thank you
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