Measuring intracellular protein concentration - (Jul/07/2008 )
I have collected 1ml E. coli culture from a shake flask. I wanted to measure the intracellular protein concentration. Therefore I lysed the cell with lysis buffer (from a DNA extraction kit, so I didn't exactly know its composition) and did a Bradford assay. However, the absorbance was very high even I diluted the sample to 1000 fold. So I am wondering whether I prepared the lysate properly. How do you guys prepare your lysate if you want measure the intracellular concentration? Can I use SDS loading buffer (without bromophenol blue) to heat lyse the bacteria and perform a Bradford assay? Thanks
I would use a standard western lysis buffer, but check the compatability with the bradford assay first. I typically use BCA assay for measuring protein concentration, it seems to cope with most lysis buffers.
Would you mind telling me the recipe of your lysis buffer?