Protein determination in a insoluble pellet - (Apr/26/2006 )
Hi, I have a question.
I have a pellet, constituted mainly of proteins, that remains after organic solvent extraction of a sample of erythrocytes previously treated with 1% Triton X-100. I want to determine the protein content of this pellet but it is very compact and I cannot solubilize it with anything. I have tested detergent solutions (Triton X-100, SDS), diluted solutions of HCl and HNO3, and Urea 6M, but I was not successful.
I do not care about denaturation, but the resulting protein solution would have to be compatible with some test of protein determination.
The original detergent insoluble pellet (before organic solvent extraction) is also very compact and it is impossible to prepare an homogeneous solution from it. Due to this fact, protein tests give irreproducible results.
Do you have any suggestion?.
Thanks to take care of my consultation.
you can solubilize in naoh and then perform the lowry protein determination. in the original lowry method, the protein was precipitated (with tca, i think) to eliminate interfering substances, solubilized with naoh then the other components were aded. this should do the trick for you.
Maybe Khejdal Total Nitrogen (times 6,25 ~ protein)?
Hi!
Try solubilizing with small amount of pellet on pipet tip in 1.5 mL eppy tube. Use 50 uL of 8M urea and allow to sit at room temp for 10 minutes. Vortex 20 sec and spin at room temp 14Krpm for 10 min. Remove supernatant and run 2uL plus 8uL of water. This will allow visualize on gel....
Then dilute with 2uL of this supernatant with 8 uL of water and run either bradford or BCA assay using dilutions of this mixture so that urea isn't in concentration to interfer.
Best of Luck!
Try solubilizing with small amount of pellet on pipet tip in 1.5 mL eppy tube. Use 50 uL of 8M urea and allow to sit at room temp for 10 minutes. Vortex 20 sec and spin at room temp 14Krpm for 10 min. Remove supernatant and run 2uL plus 8uL of water. This will allow visualize on gel....
Then dilute with 2uL of this supernatant with 8 uL of water and run either bradford or BCA assay using dilutions of this mixture so that urea isn't in concentration to interfer.
Best of Luck!
the problem with this method is you only get to know the amount of protein from the bit that you picked from the pellet. if you want to know how much protein is in the pellet then you need to solubilize the whole thing.
Thanks you people.
I didn’t expect a quick and useful answer
I’ve now a lot of work to do.
Only a question. Do you think that a I can use SDS to solublize the pellet together with the CuSO4 and Tartrate (Lowry’s method) ?. I have a protocol that use 1% SDS (final concentration) but I don’t now if this would be suitable (If this will interfer the colorimetric reaction).
Thank you again.
i believe that the cuds will precipitate and spoil the reaction (i may be wrong about this, test it by adding some of the sds buffer to the cu reagent and observe for a period of time).