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Test for protein in lysate


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4 replies to this topic

#1 Podge

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Posted 01 August 2013 - 02:18 AM

Hey,

 

I have started with my protein work to confirm some mRNA expression results with Western Blots.

When I extracted the RNA from 25mg small intestine using a Qiagen RNeasy Lipid tissue kit I kept the non-RNA phases and I now isolated the protein fraction with the "Isolation of genomic DNA and/or proteins from fatty tissue samples treated with QIAzol™ Lysis Reagent" User developed protocol.

 

I now presumably got protein in 8M Urea/50mM DTT and analysed that in a Bradford assay (had to establish that aswell in my lab...) and get concentrations of about 0.5 mg/ml; I got about 400ul of that.

 

I am just wondering if that concentration seems about right? Is there any other way to test if I actually got protein in my samples?

 

Thanks for your help!



#2 bob1

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Posted 01 August 2013 - 12:59 PM

Unfortunately for you, the Bradford assay is incompatible with more than 3 M urea and more than 5 mM DTT (see pdf here).  You could dialyse these away or precipitate the protein and resuspend in compatible buffers.



#3 Podge

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Posted 02 August 2013 - 01:02 AM

Thanks. I had looked into that and then diluted my protein solution. First 1:10 and 1:200, but neither of them showed anything above background.

So I used pure lysate at the end...

 

Will try it with precipitation and then remeasuring. thanks.



#4 Podge

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Posted 09 August 2013 - 08:18 AM

Got it working now.

 

10ul protein lysate + 70ul water + 200ul 1x dye and I get good concentrations. Blanking with the lysis buffer.Next week running my Westerns and will see what I get. Will ponceau stain the membrane to see if I get actual protein on the membrane.

 

thanks for your help!



#5 bob1

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Posted 13 August 2013 - 02:24 PM

You need to blank with lysis buffer and the water mix that you use for the sample dilution.






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