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Lysis buffer for proteins


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6 replies to this topic

#1 Darlidada

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Posted 12 January 2015 - 08:23 AM

Hi, 

 

I've some contamination on my whole protein extracts. my protein of interest is a transcription factor.

I used  Hek cells , 90 % confluent, RIPA buffer ( tris 50mM, Nacl  150mM, EDTA 5mM, NP-40 10%, Na deoxycholate 0.5% , SDS 0.1%  and water .

On my blots , i've a lot of bands, either with a validate antibody GFP. Do you have any idea about the problem ? should i change my lysis buffer , or avoid sonication ? or other ideas are more than welcome.

 

Thanks



#2 tkf

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Posted 12 January 2015 - 11:54 AM

You may need to add protease inhibitors to your lysis buffer right before you are ready to lyse. I use Roche complete tablets.



#3 bob1

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Posted 12 January 2015 - 11:55 AM

There are a number of things that could be going wrong here. Usually multiple bands on a western indicate that you haven't titrated the antibodies properly (primary and secondary) and are using them at too high a concentration or for too long an incubation period. It is also possible that you are loading too much lysate into the gel. Another thing could be that you didn't wash enough between primary and secondary antibodies or after the secondary.



#4 Darlidada

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Posted 12 January 2015 - 12:41 PM

Thanks for your answers
first, I 'm sorry I didn't precise that I'm adding the protease tablet before every use of lysis buffer.
Secondly, thanks for all these s checkpoints. I'll try to fix everything before to change the lysis buffer.
I'm still in open to other ideas.
this forum is wonderful :)

#5 GNANA

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Posted 12 January 2015 - 01:56 PM

I believe the NP40 used is 1% and not 10% as you have written.  

how does the ponceau stain on membrane look? do you able to see clear bands or kind of smear? if bands are clear may be the problem is at the blotting level, and you got to check on things that bob1 pointed.   if smeared may be you are pulling out the DNA, if not sheared properly and precipitate; that mess up right from running the gel.  i suggest you to use more lysis buffer (as you are using Hek you gonna have lot of cells) and if you choose to sonicate make sure you shear the DNA completely, else incomplete sonication itself would pull out the DNA.


I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#6 Darlidada

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Posted 12 January 2015 - 05:23 PM

Thank y ou Ghana for your reply.
You understood very well the problem. I have kind of smear with Ponceau.
I'll check on this way. In my previous lab we didn't use the sonication. And that makes me confused. I'll read about it, do you have other advices about shearing DNA ? Thanks a lot

#7 Darlidada

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Posted 30 January 2015 - 07:40 AM

It's just to close this post. I found a solution to my problem. I changed my lysis buffer (5% SDS , 5mM EDTA, 80 mM Tris ph 6.8 and 1X of tabet inhbitor) and my extraction was more clear on the blots.

Thanks for all your reply.






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