Hello everybody! I always get so good answers and help, so I'll ask you experts again. I use rat cardiac fibroblasts and do ELISA to measure cGMP. For lysis, the protocol says one can use either 0.1 M HCl or 5 % TCA followed by scraping and protein measurements and then the assay. What I find strange is that when using HCl, I get much lower protein amounts than if I use 5 % TCA (up to 10 fold less). This difference is systematic. The protein pellet is spun down at 1000 x g for 20 min at 4 degrees, and then either resuspended in 0.1 M NaOH for Bradford (coomassie plus) or TE for MicroBCA. Both assays should be compatible with HCl, especially since the HCl is aspirated from the pellet and tiny volume left is highly diluted (Bradford HCl up to 0.1 M and MicroBCA HCl up to 10 mM). I wondered first if it could be that the cells detach more easily with 5 % TCA, but looking at the wells, I cannot see any difference with my bare eyes after scraping. It is also my subjective opinion that the pellet with HCl is smaller than with 5 % TCA and that the solution is less unclear with HCl, even though the protein amount should be the same. I’m therefore not sure if it is the assay, the cell pellet or the scraping that causes this difference. Anyone that has any experience with this, or could help me explain this? Thank you for your help!
The protein is probably more soluble in 0.1 M HCl than 5% TCA, so more of the HCl-extracted protein is discarded with the supernatant.
The protein is probably more soluble in 0.1 M HCl than 5% TCA, so more of the HCl-extracted protein is discarded with the supernatant.
The pH of 0,1M HCl is 1, the pH of 5% TCA is 0,5, which may seem small but considering how low it is it may be considerable, and may precipitate more protein. Maybe adjusting HCl to 0,5M and see if it gives different results.
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