quanitation of protein extracted with trizol - (Aug/30/2006 )
I have extracted RNA and Protein from mouse organs using Trizol reagent, however I am having trouble with the quantitation of the protein, which I am assuming is due to phenol carryover. I have tried both a Bradford assay and a Lowry, but both give me very high results which are not indicative of what I actually see on the gel when I coomassie stain. I have tried loading the samples 'by eye' (looking at the band and adjusting the volume accordingly), but I can't ever seem to get an evenly loaded gel... Does anyone have any suggestions as to how I an get an evenly loaded gel? I only need 2 more westerns before I can finish my PhD - HELP!
What if you try and TCA precipitate your proteins prior to quantification?
i would do like DNA factory said.
If you can do it for next exp, try to pool your cells and divide them in 2, one for RNA and the other for protein extraction. Gives better yieds of protein and better quality too.
How does one TCA precipitate protein?
I am hoping to not have to collect anymore samples (they took over 8 months to collect) - I will most definately use a different method next time around though!
What if you try and TCA precipitate your proteins prior to quantification?
How does one TCA precipitate protein?
The procedure is the following:
1. Add 0,1 volume 1 % (w/v) NaDOC to the protein solution, mix and incubate at RT for 10 min
2. Add 0,1 volume 70 % (w/v) TCA, mix and incubate on ice for at least 15 min
3. Spin (15 min, 13.000 rpm)
4. Wash the protein pellet twice with 80 % Acetone
5. Dry pellet
The TCA acidifies high the proteins and acetone ensure precipitation of proteins.
You need to use ice cold acetone.
do not overdry your pellet. It's normal to having difficulties at resuspension step (i use buffer agitating 600rpm 37° for 1h
) thanks guys 
I'll give it a shot