Isolation of aggregated proteins

[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.