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Protein quantification after TRIzol Extraction


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#1 Yann Becker

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Posted 12 March 2014 - 03:39 AM

Hello everyone

 

I have a question about protein extraction and their quantification. First of all, sorry for my english, it's been a while since i didn't practice it and it may be a bit "rusty"

 

To quicky set the topic of the project, we are working on human platelets and we are trying to see if there is an improvement of the platelet functions after drastic weight loss (due to surgery) in obese patients. There are several protocols working quite well (agregometry, qRT PCR and such) but there is one protocols that isn't satisactory: westen blottings.

 

We use two methods to extract proteins:

- platelet lysis in Laemmli buffer (which works quite well)

- protein and RNA extraction withTRIzol (which is a pain in the glutes)

 

My problem with TRIzol extractions is that the protein yields are really random (depending on the quality of the blood samples used, the time the proteins had been stored, the alignment of the planets and some vodoo tricks i am unfamilliar with) and results in two types of blots: the over diluted ones with only faint bands and the over concentrated ones with more smear than a kindergartener's drawing (no offence, kidswink.png ).

The problem is that all the protocols i read suggests to suspend the retrieved proteins in SDS (1 to 4%) yet I have been told that SDS interfers with all the quantification tools used routinely at the lab (Qubit, Bradford Assays, Nanodrop).

 

Does anyone has any hints or tips to achieve a proper quantification for my samples?

Thank you



#2 mdfenko

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Posted 12 March 2014 - 04:12 AM

if you have some money to spend on instruments then you can try the direct detect spectrometer from millipore. according to them there is no interference from things like detergents, buffer components, etc.

 

otherwise, there are some variations to standard protein assays that are less affected by detergents (due to pretreatment of the protein mix) like this one from biorad and these from pierce.


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#3 Yann Becker

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Posted 12 March 2014 - 07:48 AM

Well since it's a lab working with state fundings, this year's fiscal exercice seems to early to spend money on new instruments (providing our unit have that much money in the first place, which i'm not quite sure) so i will probably have a look at the kits you mentionned.

Thank you






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