Test for protein in lysate - (Aug/01/2013 )
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!
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.
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.
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!
You need to blank with lysis buffer and the water mix that you use for the sample dilution.