I am acid extracting total nuclear histones from plasmodium parasites. I precipitate the acid extracted histones using 20% TCA and then washing the pellet with ice-cold acetone. The final pellet is resuspended in ultra pure water. When I estimate the protein usign BCA or bradford method the amount varies eratically between BCA or Bradford and is not matching with protein amount loaded on the SDS-PAGE (Visual estimation). Which method is best for estimating the protein concentration of histone preparations?
Hi Dhatchana!
I think performing BCA assay was a waste of time. Remember Histones don’t have cysteines, except one isoform of H3 which has only 1, they don’t have also tryptophan and can’t remember if any tyrosine. I use to work on histones but I have never carried a Bradford assay on them. A priori I think, it should work if you are working according to the instructions and within the linearity range. Remember, the quantification is relative. It is not an absolute value.
What do you mean by the quantities don’t match? Do you observe equimolar amount of the 4 histones on your gel? Do you perform nuclei isolation prior to acid extraction or you acid-extract from the whole cells? In this case, it is not an easy task to have pure histones. They are contaminated by other basic proteins such ribosomal proteins, which may co-migrate with histones.
Thanks Dr.Bobukaba for the suggestions. As you have suggested BCA underestimated the histone concentration. Bradford did work and the OD is in the linear range. I did acid extraction after isolation of nuclei of P.berghei. Even then I get some contaminating band other than five bands of histone varialnts.The contaminating bands are of high molecular weight (> 60Kda). I am working now to optimize the quality of histone prep. I would thank your suggestions in this regard.
do it at the nuclei stage, measure DNA conc in salt saturated urea then assume histones and DNA are 1:1 .. worked ok for me...
Hey Dhatchana, were u able to get rid of the contaminating band and how? I see it too..I see all my histones through ponceau but sometimes see a contaminating band too. Let me know :) I measure histones using nano drop and take the absorbance at A230 (go to activ motif website u shud be able to find out how to measure)
Dear Dhatchana, did you figure out what the best way is for total histone protein quantification? To my knowledge, Bradfold assay should work. But I have to buy the commercial kit. Did you try Sanjeevani's suggestion to measure at absorbance 230 by Nanodrop? Could you let me know if you know any easier method? Thanks a lot!
hi Dhatchana, the resulting peptides from your acid extracts can be separated using c18- reversed phase chromatography. the peptides that are eluted should immediately be ionized and inserted into a mass spectrometer, which quantifies these ions by their mass/charge ratio. the more concentrated ions ca be collided with an inert gas, and recorded in a MS/MS spectrum ( TANDEM MASS SPECTROMETER), which together with the mass/charge ratio will highlight more on the position , identification and quantity of the peptides present.
In case anyone is interested, I ran a Bradford assay (employing this reagent: Bio-Rad Protein Assay Dye Reagent Concentrate, 450 ml #5000006) doing a standard curve with BSA and Histones from Calf Thymus (10223565001, Roche) in parallel, and obtained very similar O.D. values in the two standard curves, so I think the Bradford assay works well with histones!
Best,
Irene
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