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sample sonication before loading


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6 replies to this topic

#1 biochemscientist

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Posted 20 November 2013 - 08:47 AM

Hey friends!

 

 Some of my fellows sonicate and centrifuge their extracted samples before loading into PAGE wells. I have read many papers but ddnt find any strong reason for doing this. 

 

I am doing native PAGE what do you think very mild sonication can do with my samples if i do this before loading.

 

Regards!



#2 mdfenko

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Posted 20 November 2013 - 04:12 PM

the reason for doing it with sds is to ensure that there are no particulates loaded onto the gel (sonication should disperse some and centrifugation should remove any insoluble material).

 

the danger of sonication is that you may denature the protein (mild may be okay). centrifugation should clear the sample of insoluble, denatured protein(s).


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#3 macrox

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Posted 20 November 2013 - 10:35 PM

Also, sonication shears the DNA in the sample (lysed cells). If you don't remove it either by physical methods (passing the sample though a thin needle, sonication, ...) or enzymatic methods (DNAse) the sample can be too viscous --> trouble with pipetting and loading into SDS-PAGE wells plus bad separation during electrophoresis.



#4 doxorubicin

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Posted 21 November 2013 - 01:48 AM

I've always sonicated, even "excessively", with no problems. If you don't sonicate you will sometimes have very "goopy" DNA that makes loading difficult and imprecise and the DNA will negatively affect your results. You can make a cell extract where you spin down and discard the DNA, but you should be aware that you are discarding some proteins (e.g. Histones) as well.



#5 biochemscientist

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Posted 26 November 2013 - 08:16 PM

yes i do sonicate in SDSPAGE but what about the native PAGE? where we are separating only complexes



#6 mdfenko

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Posted 27 November 2013 - 05:51 AM

again, gentle sonication may not affect your protein of interest. make sure you clarify before loading.


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#7 biochemscientist

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Posted 27 November 2013 - 07:21 PM

ok Thanks!






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