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

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

Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, please - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 10-27-2007, 07:02 PM
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Exclamation Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, please



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
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Old 10-29-2007, 05:08 AM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

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
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Old 10-29-2007, 05:12 PM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

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.
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Old 11-14-2008, 05:00 PM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

I know this message is too late for Ann Marie, but in case someone else happens upon it the problem she's having is the DNA in the lysate. That's what's causing the gel like viscosity. The simple solution is to sonicate the lysate for 10s or less, which will shear the DNA. Then the lysate will be fine for use in Westerns, etc. The 1% SDS shouldn't be a problem. I know people who lyse cells directly in Laemmli sample buffer, which is ~ 2.5% SDS and I routinely redissolve protein pellets in 2% SDS for western analysis. Works just fine.

Laura
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  #5  
Old 11-21-2008, 01:02 PM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

thanks lburger, its never too late over 2000 people have seen this and have similar issues. Who knows I may have eventually too lol... thanks for posting this.
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Old 11-27-2008, 03:04 PM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

Actually when we lyse the cells with RIPA buffer, the membranes are lyzed and the DNA comes out, which is responsible for the viscosity of the solution. the best way is to shear this DNA by ultrasonication. After sonication, centrifuge at full speed for almost 10 minutes. Might be you will not see any pellet at the bottom, but the solution will not be viscous any more.
hope it helps
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Old 04-21-2009, 10:05 AM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

I use RIPA buffer (containing 0.1%SDS) to lyse cells, but I'm wondering, would it also lyse nuclear membrane to get nuclear proteins?
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Old 04-28-2009, 09:11 AM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

I have the same problem (only with a Tris-buffer with SDS) but even after sonicating for 30 min I still cannot get rid of the DNA??? Any suggestions would be highly appreciated!! The samples are to be used for zymography so boiling etc is not an option.
Thanks
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Old 07-15-2009, 06:44 PM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

I agree with all you guys idea. I did the similar experiment with RIPA buffer, the syringe and the sonication will be good way to solve the problem. it is good to use 27G needles.
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Old 10-16-2009, 12:06 AM
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Default Re: Lysis for WB with Ripa Buffer - too viscous, no pellet - urgent help needed, plea

It's too late to help but for future reference...

RIPA will lyse nuclei.

Adding benzonase (RNase/DNase) to the RIPA is an alternative to sonication and it is gentler.
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