Has anyone measured citrate synthase (CS) activity from white adipose tissue (WAT)? I am interested in measuring CS activity from whole tissue human adipose tissue.

I am modifying a protocol that was successful in human muscle, kidney, and liver. However, due to the turbidity of the WAT homogenate we are struggling to get a meaningful read in the spetrophotometer.

At the moment I am using the following homogensing buffer:

NaCl - 150 mM

Nonidet P-40 - 1%

Tris (100 mM, pH 7.4) - 50 mM

EDTA (10 mM) - 1 mM

dissolved in MilliQ H2O and pH 7.5 (w/ HCl)

we used a 1:10 ratio (i.e., we use 10 uL of homogenising buffer per mg of WAT)

We homogenize using a TissueLyser; to remove the debris (pellet) and the insoluble fats (upper layer ring) we centrifuge at 1000 g for 10 min at 4C.

However, the middle layer that we collect as the "soluble protein" layer is still very turbid.

Ideally, I do not want to centrifuge at greater speed as we want a true "whole-tissue" homogenate, and studies are now clearly showing that spinning samples and collecting supernatant or pellet, actually introduces a bias.

any help would be very much appreciated

thanks

c