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How much pellet do you get after using RIPA? - How long to keep cells in RIPA? (Jun/06/2008 )

Hi,

I have bought a RIPA buffer from a company and have added protease inhibitor as well.

I trypsinized confluent T25 flask of HT29 and added 150ul of RIPA (I know it is going to give me a very concentrated lysate but I did that on purpose. the company suggests using more RIPA) , I centrifuges briefly to get rid of bubbles of detergents in RIPA and then vortexed briefly not to make foam, then I put on ice for 10 min.then transferred to -80 freezer directly.

today I centrifuged the samples and it gave me a lot of pellet....as much as the cell pellet after trypsinazation...I thought the lysis wasn't successful...I ran Western afterwards and got no bands....even in the control...

Do you think the cells are not lysed?

-Curtis-

Although you might have gotten no bands for other reasons such as a bad transfer (did you ponceau the membrane after transfer?), it sounds like you did not lyse your cells very well. Your pellet should definately be smaller after lysis. Normally, if all I'm doing is a western (no IP, kinase reaction, ect.) I briefly sonicate the sample (30 sec total in 10sec pulse with 30sec rest in between, on ice the whole time). If the sample is too small and you don't have access to a cup sonicator you need to pipette up and down the sample to lyse cells and shear DNA (use the 200ul). This is especially true when trying to make such a concentrated lysate. If sonicated, I immediately centrifuge and have never had issues with a large pellet. If I pipette up and down (for your sample size about 15 times should be enough) I leave the tube on ice for about 10-15 mins and then centrifuge. If a large pellet remains I'll pipette more, respin. If you still have samples in the freezer, when you thaw them you can pipette/sonicate, recentrifuge and they should be ok. Maybe you should have someone in your lab show you how to do a Bradford assay. This is a quick method to measure the protein concentration in your cell lysate. This way you know how concentrated your lysate really is. This also allows you to know how much protein you actually ran on the gel.

-rkay447-

QUOTE (rkay447 @ Jun 7 2008, 05:18 AM)
Although you might have gotten no bands for other reasons such as a bad transfer (did you ponceau the membrane after transfer?), it sounds like you did not lyse your cells very well. Your pellet should definately be smaller after lysis. Normally, if all I'm doing is a western (no IP, kinase reaction, ect.) I briefly sonicate the sample (30 sec total in 10sec pulse with 30sec rest in between, on ice the whole time). If the sample is too small and you don't have access to a cup sonicator you need to pipette up and down the sample to lyse cells and shear DNA (use the 200ul). This is especially true when trying to make such a concentrated lysate. If sonicated, I immediately centrifuge and have never had issues with a large pellet. If I pipette up and down (for your sample size about 15 times should be enough) I leave the tube on ice for about 10-15 mins and then centrifuge. If a large pellet remains I'll pipette more, respin. If you still have samples in the freezer, when you thaw them you can pipette/sonicate, recentrifuge and they should be ok. Maybe you should have someone in your lab show you how to do a Bradford assay. This is a quick method to measure the protein concentration in your cell lysate. This way you know how concentrated your lysate really is. This also allows you to know how much protein you actually ran on the gel.



Pipette up and down abt 15 times.....is it ok because this will create many foams/bubbles......

-sasoriza-

QUOTE (rkay447 @ Jun 7 2008, 06:18 AM)
Although you might have gotten no bands for other reasons such as a bad transfer (did you ponceau the membrane after transfer?), it sounds like you did not lyse your cells very well. Your pellet should definately be smaller after lysis. Normally, if all I'm doing is a western (no IP, kinase reaction, ect.) I briefly sonicate the sample (30 sec total in 10sec pulse with 30sec rest in between, on ice the whole time). If the sample is too small and you don't have access to a cup sonicator you need to pipette up and down the sample to lyse cells and shear DNA (use the 200ul). This is especially true when trying to make such a concentrated lysate. If sonicated, I immediately centrifuge and have never had issues with a large pellet. If I pipette up and down (for your sample size about 15 times should be enough) I leave the tube on ice for about 10-15 mins and then centrifuge. If a large pellet remains I'll pipette more, respin. If you still have samples in the freezer, when you thaw them you can pipette/sonicate, recentrifuge and they should be ok. Maybe you should have someone in your lab show you how to do a Bradford assay. This is a quick method to measure the protein concentration in your cell lysate. This way you know how concentrated your lysate really is. This also allows you to know how much protein you actually ran on the gel.


yep........thank you very much for the full explanation

I will run a Bradfor Assay tomorrow. thanks again

-Curtis-

It's fine because you need to centrifuge your samples afterward to pellet the chromatin. During the centrifugation you loose all this. It's ok to have some bubbles and foam in your sample. You just don't want your entire sample foamed up. If you are careful and don't take the entire sample up into the tip, only about 3/4 is enough, and don't take the tip out of the sample when you pipette back down, you won't create as much as you think.

-rkay447-