zhangyin777

Hello everyone I would like to detect the aggregation of newly synthesized proteins, but in the process I have encountered some problems. My strategy was to label the newly synthesized protein in vivo with 5ug/ml puromycin for 30 min, then through the differential centrifugation to isolate the aggregated proteins. Finally, anti-puromycin were used to detect newly synthesized proteins in aggregated proteins. Question 1. I found that each time I do this experiment, it was difficult to keep the same amount of aggregated proteins after centrifugation. I mean that there were sometimes more or less aggregated proteins in the same strain. Is there any way to ensure that each operation is as same as possible? What are the cautions for this experiment? Question 2. I used puromycin antibodies to detect newly synthesized proteins in aggregation. The same strain sometimes has more total aggregated proteins, of which there are fewer newly synthesized proteins, but sometimes the results are opposite. Is there any possible reason? Thank you very much! Ps. Total is total proteins, Pellet is aggregated proteins.

Hello everyone I would like to purify the newly synthesized protein by the biotin-streptavidin system, but in the process I have encountered some problems. My strategy was to label the newly synthesized protein in vitro with biotin-puromycin, then bind the labeled protein with streptavidin-beads, and finally elute with 8M guanidine hydrochloride, pH 1.5. After I get the protein, I need to perform iTRAQ. analysis. Question 1. When I detected the biotin-puromycin-labeled protein by western blot, I found that there are many proteins around 40kD. They may bind too many streptavidin-beads during the purification process, causing other proteins to fail to bind to beads, this may affect subsequent iTRAQ analysis. I wonder what these proteins might be? How do I eliminate or reduce the effect of these proteins on the purification process? Question 2, My purification system is very inefficient, and the elution yields few proteins that can't satisfy the subsequent iTRAQ analysis. My current binding buffer and wash buffer are PBS. The elution buffer is 8M guanidine hydrochloride, pH 1.5, but why the protein detected in elution is very weak. What could be the reason? Thank you very much!