Hi, I previously assessed enzymatic activity of SOD1 from mice spinal cords homogenates with great success. Basically, I homogenized proteins in TNG-T buffer (50 mM Tris–HCl, pH 7.4/150 mM NaCl/10% glycerol/1% Triton X-100/protease inhibitor mixture) and spun at 1000g for 10m. I did not denaturate the samples at any time and I used non-denaturing gradient gels for the enzymatic activity assay. The article in which I presented this data: http://www.ncbi.nlm.nih.gov/pubmed/20573565

Hi. Maybe it depends on which organic components you want to extract during brain tissue homogenization and centrifugation process (eg. hydro-alcoholic medium, maybe?). Another crucial point could be represented by reagent concentrations for the antioxidant assays you mentioned: try to increase (or to correct) the concentrations of the analytic reagents to improve the antioxidant resolution

For LPO, you have to collect homogenate without cell debris (by centrifuging your crude homogenate @2500 rpm for 5 min).

We worked on 5% homogenate that works very well. may I know how old are your sample? did you use proteinase inhibitor in your buffer cocktail? Fresh samples are always preferred. Always use proteinase inhibitor to avoid degradation of proteins. Supernatant@10000 rpm is good for enzymatic assays like CAT, GST, GPx, SOD, GSH.

Probably, you have high content of enzymes in 10% homogenate or PMS, so you are not getting the any significant results. Try 5% homogenate, store at 20C and use aliquot for each assays. The buffer temperature is also important. The assay buffer temp must be 37 C during assay (avoid putting substrate on 37 c). All the best.

@ Kumar Vaibhav. Thank you. But most of the papers use 10% homogenate? So in my case, do you think a 5% homogenate will help?