What exactly do you mean by blank? I suppose you are referring to control (which contain everything with the exception of enzyme or with heat killed enzyme).

Blank truely speaking is the one with which you set zero absorbance in spectrophotomter. Kindly understand basic principle behind the assay (how are superoxide anion radicals, the basic substrate for SOD, are generated, etc.).

Control value has to be higher than that of assay samples.

Respected Prof. P. Pardha-Saradhi,

                    I was referring to control.Thanks a lot.

for measuring SOD, we used "two" blanks:

- the first is to set spectrophotometer zeroing and it is containing buffer with all components expect sample (yellow color

- the second is the same tube used in blank 1 but after light expostion. this is considered as 100% of NBT reduction due to radical. indeed in samples' tube OD will be lower as activity is calculated based the % of inhibition of  NBT photoreduction

For SOD assay: BLANK contains all components. According to principle superoxide radicals are generated in PMS-NADH systems by oxidation of NADH and assayed by the reduction of NBT. CONTROL contains BLNK+PMS. SAMPLE contains CONTROL+enzyme (your sample/plasma). Decrease absorbance of the reaction mixture in plasma indicate increase superoxide anion scavenging activity.

dear,

thank you for your question, the blank is all components (blank+PMS) so the decreased absorbance means the increase in activity of SOD.

so from high blank to less.

best regards

NOT IN ALL CASES WILL THE CONTROL SOD ACTIVITY BE HIGHER THAN THE TEST SOD ACTIVITY. YOU NEED TO UNDERSTAND THAT INITIALLY, SOD ACTIVITY WILL RISE IN RESPONSE TO ROS GENERATION, AND SO IF YOU ASSAY FOR THE ACTIVITY AT THAT STAGE, IT WILL BE HIGHER THAN CONTROL.  BUT PROLONG AND CONTINUOUS GENERATION OF ROS WILL OVERWHELM THE ACTIVITY OF THIS ENZYME, THUS, THE ACTIVITY WILL BE LOWER THAN THE CONTROL. THIS APPLIES FOR ALL THE OTHER ANTIOXIDANT ENZYMES. 

Superoxide dismutase catalyzes the dismutation of the very active oxygen species  superoxide anion into molecular oxygen and the less harmful hydrogen peroxide, and this reaction provides a very important defense against oxidative damage.  The most commonly used method to measure SOD activity is by the inhibition a reaction dependent on superoxide anion where superoxide ions are to be generated first, say from the reaction between xanthine and molecular O2 in the presence of catalyst xanthine oxidase to form two more products in addition to the superoxide anion and these are uric acid and hydrogen peroxide. Then another reaction is to be initiated say a substrate that easily gets reduced after the reaction with the newly formed superoxide anion, like tetrazolium salt (nitroblue tetrazolium, NBT) that is being converted into a formazan dye (NBT-diformazan), the rate of this second reaction can easily be monitored using a spectrophotometer that measures the amount of the colored NBT-diformazan dye product. Addition of SOD into this reaction therefore reduces superoxide ion levels, thereby lowering the rate of formazan dye formation. SOD activity in the experimental sample is measured as the percent inhibition of the rate of formazan dye formation. In this case there a few blanks to prepare--sample blank (no SOD), reagent blank (no enzyme solution for the first reaction generating superoxide ions, no xanthine oxidase), and the combination of sample and reagent blank. The blank that does not have the SOD extract sample is expected to have higher absorbance since more colored formazan dye will be produced. The one with SOD extract sample will inhibit the formation of formazan dye product and a 50% inhibition is equivalent to 1 unit of SOD. You may search the Internet for more related work.

Normally, in SOD determination kits, 50% inhibition activity of SOD or SOD-like materials is determined by a colorimetric method. Since absorbance is proportional to the amount of superoxide anion, the SOD activity is quantified by measuring decrease in color development. The blank will give higher absorbance than the tube containing SOD extract. Reducing agents like ascorbic acid and reduced glutathione interfere with SOD assay.

if you use NBT method the color will decrease with increasing SOD

Your Control or Black will have high absorbance.

In thecColorimetric method for SOD determination using  decrease of superoxide anion absorción at 420 nm, the blank should have higher absorbance than the sample containing SOD.

Of course the control will have a higher reading as Evangelina S Ella  has explained and as the definition of SOD express.

•  Use blank with all reagents except sample in mixture and read at desired wavelength, you will got high value of blank at every cost if no expermental error in your work. chill and enjoy

Please have someone succeeded in developing SOD assays without a test-kit and how did they manage to get around with hypoxanthine solubilityu. I'm currently working on an developing one but I have serious issues with Hypoxanthine because its not soluble and does not give a good calibration. could xanthine be a better option?

what will by the formula to convert %age inhibition to units if i got 70% or 90% inhibition what will be the units of SOD. and these units/ml of mixture used or 10 or 20 ul or 1ml etc