Problem concerning protein quantitation - (Mar/24/2009 )

Hi, I use the Bradford method to measure protein concentration.

I use three replicates for each standard points and there are in total 6 standard pts I use (from 10 to 35 microgram per ml).

However, i discover that at the point 20 and 25 microgram per ml, the absorbance at 595nm is always not that consistent. Other points are absolutely fine and I have pipette cautiously and ensure that everything I added is correct. Does anyone know why is this so?

By the way, may I know which brand of Ovalbumin ur lab is using?

Thx in advance.

-barnacleman-

Hey,

I am not a gr8 fan of bradford for protein estimation. I prefer using BCA and the best is lowry. These assays are more sensitive. And I use the BSA which comes as a part of the assay kit. Otherwise weigh the sigma BSA for making standards.

Hope it helps

Best,
TC

barnacleman on Mar 25 2009, 10:29 AM said:

-T C-

barnacleman on Mar 24 2009, 11:59 PM said:

you pipette cautiously but is your pipette properly calibrated?

are you using the same pipette for the 20 and 25 ug/ml standards that you use for the other standards?

are those values near or at the lower and/or upper limit of the pipettes' range?

-mdfenko-