Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, please

[Anne-Marie]

Dear All,

I was doing a lysis of my cells in order to do a Western Blotting later on.

I had my cells in 6-well plates, highly confluent, and used Ripa Buffer with 1% SDS (I believe I was supposed to have used 0.1%)

After scrapping and centrifuging, I still had no pellet, and the solution was just as viscous as before. I repeated the centrifugation at 4C and still no pellet was formed.

I was wondering if you know what happened (for usually I get a normal pellet), and most importantly, how the samples could still be recuperated (they are frozen now).
They were from a very important experiment….

Thanks a lot!

Anne-Marie

[admin]

Hello Anne Marie,
what is the protein you are trying to isolate? Is it membrane bound or cytosolic?

The problem may have been the high SDS. As you know SDS is a detergent and may have damaged your cell membranes. Thus when you centrifuge you aren't getting a pellet because the membranes are broken up.

You could try running as much sample as you can on a gel and conduct western blot.
You may assess the quantity of protein to load by doing a protein assay. That way you will know if there is enough to see anything.

Usually when I scrape my 6-well plates I never get a pellet ( I use lysis buffer to scrape), and the pellet is cell junk that I throw away anyways. Not sure if you are scraping for the cells, or lysing.

Your Ripa Buffer with 1% SDS is that a lysis buffer? if its just for scraping, I would add extra buffers to make it a lysis buffer and then use a syringe to pass the samples through the needle to lyse fully.

Even if your samples are not that concentrated ( by protein concentration determination), you could easily do protein concentration methods such as vacuum, lyophilization and other protein concentration methods.

Anybody have better ideas?
cheers

[danfive]

The high SDS lysed your cells. So you just have a protein solution. Centrifuge at high speed (14,000xg) for 10 minutes (don't expect a pellet) and you can use the supernatant for your downstream applications. I'm guessing your 6-well plates are like some I've seen, these usually don't contain a lot of cells, as in enough to produce big pellets of cell debris anyway.