Is there any difference in protein content or alteration in phosphorylation of protein by using scrapping method and trypsin method for doing western blot?
In contrast to previous answers, it DOES matter if you scrape or trypsinize. Many groups have performed these types of analyses comparing the difference in cell viability and metabolic parameters in cells that were trypsinized or scraped (here is one, PMID: 20337597). Many have found that trypsinizing causes less death compared to scraping, as well as causes fewer changes in metabolic parameters (most likely because most of the cells are still intact). In contrast, trypsin is a protease and if you are examining membrane spanning or membrane associated proteins, then this will definitely affect your assay and readout.
So overall, you cannot make a blanket statement that there are no differences between the two. Maybe for some proteins, no noticeable differences are observed, but this is not the case for every protein, cell, or situation. You need to evaluate the pros and cons in your system, and make the correct decision on which way to pursue the assay.
Good luck,
~Adam
Dear Asiya,
Scrapping and trypsiniing cells is a quick procedure, i don't believe this should affect your desired protein. Which protein are you interested in detecting?
Dear Asiya,
It makes no difference either by scrapping or trypsinizing cells for protein extraction. Both the procedures are done for different purpose
1. Trypsinisation: If you want to count the cellsand to look for viable cells eg., Trypan blue dye exclusion test to check for dead cells after treatment
2.Scrapping: you may prefer this in case if you want to study the phosphorylation status of certain kinases at smaller time intervals ( eg. 5, 10,15, 20 minutes following drug treatment). In this case you can directly add the lysis buffer with phosphatase inhibitors to the plate and collect the lysed cells by scrapping and proceed for protein quantitation and PAGE.
In contrast to previous answers, it DOES matter if you scrape or trypsinize. Many groups have performed these types of analyses comparing the difference in cell viability and metabolic parameters in cells that were trypsinized or scraped (here is one, PMID: 20337597). Many have found that trypsinizing causes less death compared to scraping, as well as causes fewer changes in metabolic parameters (most likely because most of the cells are still intact). In contrast, trypsin is a protease and if you are examining membrane spanning or membrane associated proteins, then this will definitely affect your assay and readout.
So overall, you cannot make a blanket statement that there are no differences between the two. Maybe for some proteins, no noticeable differences are observed, but this is not the case for every protein, cell, or situation. You need to evaluate the pros and cons in your system, and make the correct decision on which way to pursue the assay.
Good luck,
~Adam
I believe the location of your protein is very important to decide the method for protein extraction. Where is your protein located?, Is it on the cell membrane??
Thanks for your reply. I am going to see the P-Akt and P-p38 . So they are not membrane proteins. I am going to try trypsin method.
Thanks Adam for your suggestions, I totally agree with your points to be considered before choosing trypsination or cell scrapping.
I prefer to scrape cells in the plate in lysis buffer. Cells will die from scraping, but they have to be lysed anyway. I think this way you stop all the processes that are going on in the cells almost instantly. You don't know what trypsinisation is going to do to these processes.
In addition, to block trypsin you normally add medium containg FCS. This may influence processes in the cells (in this case I'm thinking of the mTOR pathway, for example).
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