Hello,

I`m quite new in the world of science, so my questions might seem a bit stupid:)...but I hope that you will forgive me and most importantly, I`m counting on your help:)

I`m growing Hela cells and transfecting them with protein expression plasmids and after transfection I want to do Western to detect protein expression and I also want to isolate RNA.
I don`t want to use Trizol for protein isolation and because of that I can`t lyse the cells directly on the plate with Trizol.
So I decided to just trypsinize the cells, collect them, take an aliquot for Western (then pellet the aliquot and resuspend in lysis buffer) and pellet the rest of the cells and resuspend in Trizol and store in -80C until I`m ready for RNA isolation. But then I was told that this way I will not get a good quality RNA and the quality of RNA is very important for me. So then I washed the cells with PBS and put on PBS-EDTA and after 10 minutes or even more, I started washing off the cells. It wasn`t the easiest job as they were „holding on” to the plate quite strongly. At some point it seemed to me that the plate was clean (but unfortunately in my own stupidity I didn`t check under the microscope) and I stopped. I took and aliquot for Western and the rest of the cells I resuspended in Trizol. I got a lot less total protein compared to when I detached cells with trypsin, but I also got a better Western, which means that I got more exogenous protein. But the amount of RNA was also a lot smaller, it was too small and I thought that I probably didn`t get all the cells from the plate as they were attached so strongly. So the next time I decided to try scraping the cells. I scraped them and collected them in PBS and again took an aliquot for Western and stored the other cells for RNA isolation. This time I checked under the microscope and was very happy because the plate was clean, but then I was told that by scraping I could damage my cells so much that I will loose most of the important “things” into PBS. Now, I have done Western again and it was ok, maybe even better than last time, but to be honest I was hoping to get a lot more total protein, so I`m a bit worried, that maybe I DID damage my cells a lot. I haven`t been able to isolate RNA yet, so I don`t know how much I will get it this time.
But anyhow, my questions are
- Does anyone detach Hela cells without trypsin? And how?
- Does anyone use accutase or alfazyme for detaching cells? I`ve read that they are more gentle than trypsin.
- What`s the story with scraping - If it damages cells so much then why is it used? Or is there some kind of trick to avoid damaging (Is scraping in PBS ok? Should I scrape on ice? How much buffer should I use when scraping cells from 10-cm plates? etc.)

I hope you can give me some good ideas.


Thanks!

Ok, there are two real problems here: 1) separate extractions of protein and RNA and 2) changes in expression of protein/RNA by trypsin or other methods of harvesting.

1) If you really need to do both protein and RNA, set up separate wells of a 6 well plate, seeded and treated at the same time points from the same stocks and use one set of wells for protein and one set for RNA.

2) Depending on your protein, harvesting using trypsin may not have much of an effect - e.g. if your protein is intracellular. However, if your protein is involved in the membrane or extracellular matrix, or even involved in the manufacture or transport of cell membrane or ECM proteins, then trypsin may have an effect on what you are seeing.
Scraping on the other hand, will damage pretty much all the cells by mechanical forces. It is quick and easy so there may not be changes in expression over your harvesting time, but you may lose stuff in wash steps. To prevent this you could just wash your cells on a plate/dish/flask and then scrape into a tube of lysis buffer, or even lyse in the plate with no scraping involved, other than when all the cells are lysed and you are transferring to a tube.

The method of harvesting that you should choose is the one that will cause the least damage to the pathway(s) that you are looking at.

Inactivate the trypsin after harvesting with medium containing serum. Aliquot your sample, spin your cells, wash them once/ twice with PBS buffer and froze/lyse you cells with your buffer.

I think there is no problem for protein samples from tripsinized cells in WB (trypsinization should be stopped before lyse), because I also do that way sometime. And my advisor told us at any time protein should be collected directly by SDS-loading buffer for WB, unless some specific usage (kinase assay, IP,or membrane protein extraction). Using RIPA or PBS to collect protein would cause a great lost of membrane or organelle protein. May it help you. Good luck.