Cell lysis before measuring total protein content - Which method for lysis can be combined with the Bradford method? (Jan/25/2006 )
First, I apologize if this is something that has been discussed earlier in this forum; if so, give me a hint about where to find the information.
I need to lyse some cells (HEK293) to measure total protein content by BioRad (Modified Bradford Method). Does anybody have a protocol for this? The extract will not be used for any downstream applications after the protein measurement.
It will be of importance to get hold of as many proteins as possible, including relatively insoluble lipoproteins, which probably means that I should use some kind of detergent (Triton x-100 or SDS).
Any hints are greatly appreciated
Cells can be broken open (lysed) to liberate their contents using a variety of methods. The most used method is detergent lysis, in which cell membranes are solubilized using a detergent. A variety of detergents are commonly used. These detergents differ in their chemical structure, charge, and capacity to solubilize membrane and cytoskeletal proteins.
Lysis buffers contain a variety of chemicals in addition to detergent. For instance in the RIPA lysis buffer contains more than one type of detergent :
component concentration function
IGEPAL CA-630(NP40) or Triton X-100 1% detergent (solubilize membranes)
sodium deoxycholate 0.5% detergent (solubilize membranes)
SDS 0.1% detergent (solubilize membranes)
Unfortunately I could't find again where I saw that but I once read that Triton X100 absorbs light at 595nm so it is not not recommended when you want to measure ptotein concentration.
For that reason I always use NP-40.
But you should actually test in your protein lysates what is the best.
I actually lysed HEK 293 cells in :
50mM tris-HCl pH 7.5,
it worked dine for the proteins I was interested in.
You just have to fins out.
I use a buffer similar to the one mentioned by macedo. however, it still interferes with the O.D. measurements. therefore, I add the same volume of buffer as the sample volume, to the BSA calibrations and this actually includes the interference in the reading.
BTW, in the link below you will find a table (page 7) which includes all the reagents compatible with the method, and it is stated there that NP-40 is compatible only up yo 0.25%
Technical question - is it really necessary to add sodium deoxycholate to the lysis buffer? Isn't SDS sufficient to solubilize the membranes?
as far as I know any lysate by any common lysis buffer can be measured by Bradford.
we just observed yesterday that if we add too much protease inhibitor to our lysis buffer it'll interfere with our results.