Odds, Ends, Protein Concentrations, and CAT Standards

CAT KILLER STRIKES AGAIN! My latest victim bore the additional burden of the lowest protein concentration of any transfected samples thus far. Could this be due to the room temperature lysis buffer incubation I had performed? I’m not sure. I would be able to narrow down possible causes of this low protein concentration had I done what was suggested (take a 10 microliter portion of one of the samples after the thirty-minute lysis buffer incubation and observe whether or not the cells looked whole). I forgot this step in a rush to determine protein yield with high hopes that this would be my first successful CAT ELISA with a high protein yield due to the room temperature lysis conditions. Since CAT ELISAs depend on cell lysis for effective measurement of protein concentrations, whole cells appearing after the incubation with lysis buffer would lower measured protein concentrations. Subsequent centrifugation of whole cell samples would cause high levels of protein loss as the protein would pellet out contained in the cell. Instead of wasting plasmids on a process that my or may not work, the next step is seeding normal HeLa cells in plates and subjecting different plates to room temperature, ice and half-room temperature/half-ice lysis to determine which conditions are least conducive to whole cell state maintenance. Another recent critical change to the protocol is the use of 10 microliters of thyroid hormone rather than the one microliter amounts previously used; if I ever proceed beyond the Bradford Assay again, this larger amount of thyroid hormone will hopefully solve the issue of no apparent thyroid hormone induction. Unfortunately, I’m also still getting exponential CAT Standard curves where they should be linear. I’m not sure how to fix this problem besides trying to epitomize vigorous pipetting to ensure uniform CAT Enzyme distribution.

On the upside, there have been far fewer incidences of contamination this summer as compared to last summer so there are fewer delays in the form of waiting for new and uncontaminated flasks to become confluent. For now, this the host of ‘How not to do a CAT ELISA’ signing off.