I have tried to estimate SOD from blood hemolysate by Pyragallol autooxidation method of Marland n Markland.
I have followed the following steps :
BLANK: 50mM Tris EDTA Buffer. (ph-8.5)
Control: TE Buffer + Dist.water+ Pyragallol
Sample: TE + hemolysate + PG.
Observed for 5 mins at 420nm for 5 mins.
But I am getting higher sample Absorbance above the control. I tried diluting till 100x n 500x. But the colour of the hemolysate Is not allowing the proper estimation of SOD percentage inhibition.
I think I am making an error with the grouping. Can anyone suggest me a way to overcome the background influence of the hemolysate using this same method.
you can get rid of the color by treat the blood samples with chloroform-ethanol after separation of the erythrocytes from the blood and wash them
then add the solvent mixture, the SOD will be present in the clear top layer.
you can find this method in Winterburn et. al. paper published in 1975 ,in J. Lab. Clin Med ; Feb. :337-341. one of my students tried it it worked nicely.
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