I have the Abs. values and i want to convert it to mg/g FW or any other unit, the reaction mix was riboflavin, L-methionine and NBT.
Assay of superoxide dismutase activity by combining
electrophoresis and densitometry
Ching-Nen Chen and Shu-Mei Pan1
Department of Botany, National Taiwan University, Taipei, Taiwan 10764, Republic of China
(Received November 20, 1995; Accepted March 2, 1996)
Abstract. A modified technique was developed to assay superoxide dismutase (SOD) activity by combining polyacrylamide gel electrophoresis and densitometry. After electrophoresis on native polyacrylamide gels, the negative banding corresponding to the SOD activity was visualized by soaking the gels in nitroblue tetrazolium then riboflavin, and finally exposing to light. Effects of the banding of SOD activity induced by different soaking durations and light intensities were evaluated in this system. The optimal soaking duration was determined to be 15 min for each of the two soaking steps, while the optimal exposure was 30 mEm-2s-1for 15 min. The gels were then immediately scanned with a laser densitometer, and the readings of the samples corresponding to their total SOD activity were obtained by processing the image. A standard curve was prepared with a serial dilution of partially purified SOD, whose activity was previously determined by using a spectrophotometric method. The total SOD activity of an unknown sample could be obtained by interpolating its reading to the standard curve. The activity of a single SOD isozyme of a sample could also be obtained with the same procedure. The technique was ten times more efficient than the spectrophotometric method. The interference coming from non-SOD substances in the crude extract could be removed by electrophoresis. The standard deviations of the SOD activity of the crude extracts from rice seedlings, papaya, and tobacco leaves measured with the technique were less than 9%, 7%, and 8% (for each n = 6, on 6 gels), respectively.
http://ejournal.sinica.edu.tw/bbas/content/1996/2/bot372-03.html
thank you Dr. Ali
but i only have Absorbence values of SOD
I just need the equation to convert that into activity
Estimation of superoxide dismutase activity:
Superoxide dismutase activity was determined according to Nishikimi et al. (1972).
Reagents:
A- 50 mM phosphate buffer at pH 8.5
B- 1 mM nitroblue tetrazolium
C- 1 mM NADH in the buffer A
D- 0.1 mM phenazine methosulphate
E- Superoxide dismutase (the enzyme extract)
Procedure:
A working reagent was firstly prepared by mixing reagent A, B and C in a ratio of 10: 1: 1 ml. The following solutions were then pipetted:
Reagent
Sample
Blank
Working reagent
1 ml
1 ml
E
0.1 ml
-
Distilled water
-
0.1 ml
D
0.1 ml
0.1 ml
The contents of each of the sample and blank were immediately mixed by inversion and the increase in absorbance (rA) at 560 nm was recorded for approximately 5 minutes for both of the sample and blank. The enzyme activity (Unit ml-1) could be calculated as following:
Enzyme activity = [(rA560 blank - rA560 sample) /
rA560 blank] X 100 X 3.75
thank you Dr./ Heshmat
that was too helpful
but you mentioned that the final volume is 1.2 ml, while i used 3 ml final volume
does that change anything in the equation.
thank you
Dear Heshmat Soliman Aldesuquy ,
What is 3.75 in the equation? Is that the volume You have used or it is some kind of constant?
Thanks
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