I have a query regarding estimation of axonemes using colorimetric assay
(Bradfords assay) I have extracted flagella from the wild type and some pf
mutants. I have demembranated the flagella with NP40 (0.5%). I resuspended
the axoneme pellet(extracted from 1-1.5L of mid-log phase culture) in 300 ul
of HMDEK buffer after washing thrice with the 1 ml of same buffer (without
NP40), and tried to estimate the axoneme using bradford assay, I find that
my axoneme containing solution is not homogeneous(looks turbid). Under
microscope, the axoneme looks fine without any contamination. How do I use
this for protein estimation. I tried estimating the proteins as it is and
diluted but the content was too low in both the cases. I also added 6M
Urea with Betamercaptoethanol (to solubilize the proteins) to the axonemes
and then used it for estimation but that too did not work in fact the OD of
axoneme was lower than the blank and in some cases I see large deviation in
the readings(in the duplicate and triplicate samples). I'm using ready
to use Bradford reagent from Fermentas for estimation, which
is compatible with 0.5% of NP 40 so detergent interference should not be an
issue. I need equal loading of the WT and pf mutants flagella for my
Westerns. How do I solve this problem?
Any help appreciated..
Thanks and Regards
Rao Venkatramanan G.
Mumbai University campus,