I am isolating histones using 0.4N H2SO4. I am using 1000000 cells and dissolving the desired histone pellet in 100 microlitres of Milli Q water with 0.1mM PMSF. Although I am getting good preparations of histones but I am not able to quantify the histones at 230nm. I need to proceed ahead with western blotting where i need to load equal quantity of protein. Kindly suggest what can be done?
Perhaps you need to use a different wavelength. You could also run some of the sample (20-30 micro litres) on SDS-PAGE, along with 1 microgram of BSA. Stain with Coomassie Brilliant Blue R250 and compare the amount of histone to the BSA standard.
Use another simple and quick prot determination method: bradford, bicinchoninic acid
they should Work well and are quick to perform, and are more "quantitatve" that 230 detn
i hoeever think that if equipment is well calibrated you should see absorbance of proteins at 230 and,or 280 nm. I never worked with histones but i would asume that they have aromatic amino acids, i mean amino acids that absorb in UV or that react with bradford reagents
I have tried more or less everything to quantify histone protein. But nothing actually works in the perfect sense. Best you could do as suggested by Bruce. But I would suggest to use silver staining instead of using Coomassie Brilliant Blue R250 as they always are in less quantitiy so really precious.
Electrophoresis is the last resource as is a qualitative procedure (sure you may play around to do it semi quantitative)
I have figured out that Histones do not absorb much in the near UV region. Also that BCA should not work due to histones amino acid composition.
Also apparently, Bradford should work . Also the "vintage" Lowry method should also work. But be sure that you do not use the usual proteins as standard. You should use histones as primary standards
It seems that better than Lowry (for histones) is a method I never used: the reineckate acid method
On the other hand, I think these 2 books and these 2 papers may be helpful
Chromatin
Escrito por Kensal E. van Holde
Methods of Biochemical Analysis, Volumen 14
editado por David Glick
Preparation of histones by the use of Reinecke salt. Analytical Biochem
Nils Olof Lindh, Bo L. Brantmark
Analytical Biochemistry
Volume 10, Issue 3, March 1965, Pages 415-420
Influence of carbamoylation on some analytical properties of basic polypeptides.
Lea MA, Luke A, Martinson C, Velazquez O.
Int J Pept Protein Res. 1986 Mar;27(3):251-60.
Thanks all,
I have used bradford method to estimate histones. It has clicked well.
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