Hi Lara,

We usually used kinetic measurements and thus calculated Vmax as a measure of SOD activity.

Anyway, the best approach if you have a calibration curve by known SOD activity. Moreover in the datasheet to the mentioned kit used 3 different blanks to calculate inhibition %. So, it would be easier to help if you provide more data on your measurements.

https://www.abcam.com/ps/products/65/ab65354/documents/ab65354%20Superoxide%20Dismutase%20Activity%20assay%20v9b%20(website).pdf

Hi, thank you for your answer. Yes, there are three blanks, which I have done. They are used to calculate inhibition, but when I do the calculation, results make no sense, because they are over 100%

For example: Blank1=0,152

Blank2=1,259

Blank3=0,107

Sample=0,468

When calculated as recommended in the protocol I get 1857,7% This is one example, but all samples have a similar issue.

Any idea why that could be, and if there is any simpler calculation I could try? Again, I just need to find out if SOD activity was lower in my control samples or not.

Thanks again :)

Hi Lara,

Are your results reproducible? If you get the same results each time it could be related to the nature of your sample and used kit limitations.

Kit based on the coupling of two enzymatic reactions - Xanthine Oxidase (XO) as a O2* generator and WST-1 as an acceptor of the free electrons. Of cause it is a simplified description and you could find more details by the following link.

Article Measurement of superoxide dismutase, catalase and glutathion...

So, if your sample contains XO or suitable substrates, other electron donors (NADH, NADPH etc.) or molecules affecting coupling of reactions it could affect WST-1 reduction a lot and thus result in formazan accumulation and increasing of optical density. In my past, I saw it once in undiluted fresh blood lysates (immediate MTT reduction).

You reported OD for your sample (without XO) 1.259 what looks good enough for measurement by modern instruments. At the same time, you reported for XO blank1 only 0.152 what is really on the bottom of the quantification limit for OD measurements. It tells what is me something wrong with the setup (b1 and b2 substitution will give 72 %). XO sample should give you the highest OD and should be optimized to be around 1 OD for optimal incubation time for the endpoint scenario.

To address the problem I would check all positions and names first (reproducibility) and check different dilutions of your sample (effect of dilution).

Oh, I forgot about Thermo fisher - did you asked support about the mentioned problem?

And do not forget some other SOD assays available f.e. we used 2 different systems based on adrenaline hydrochloride and quercetin (XO recognized as the best).

Hopefully, it will help!

Daniil

Hello!

What are your samples? What is it (serum or blood plasma, something else)?

Yes, absorption levels (optical density) can be used to compare enzyme activity in samples.

This is usually done in two ways:

1.The optical density is measured at some point in time (T = 0), and then it is measured after a certain period of time has elapsed (after 1, 2 minutes or more). The difference in optical density between these measurements will characterize the activity of the enzyme (the smaller this difference, the higher the activity of the enzyme and vice versa - the dependence is inversely proportional)

2. measure the optical density in a continuous mode (more precisely, at certain intervals between measurements) for some time). I do this myself when I need to measure the activity of an enzyme.

If the enzyme activity in your samples is higher than that in the control sample, then the difference in optical density for the samples should be less than for the control sample. And vice versa.

It's not entirely clear why you have such different meanings for blanks?

Are these different blanks or are they the same, but measured 3 times?

What is the expiration date of your kit? In what conditions was it stored?

Maybe you should try other methods of detecting activity?

For example: https://www.researchgate.net/publication/344319356_Determination_of_superoxide_dismutase_activity

DOI: 10.13140/RG.2.2.32125.18400

Hi, thank you for your answer!

It was an insect tissue, homogenized per instructions in the protocol.

Could you please elaborate on the first method a bit further? Do I expect the absorbance to increase or decrease with the time? For example, if absorbance at T0 was 0.250, and five minutes later 0.253, do I note it as +3. What if in the case of some samples it decreased? Do I take it as a negative value then?

Those are three different blanks.

Blank 1 = H2O, WST, and enzyme

Blank 2 = sample, WST, and dilution buffer

Blank 3 = H2O, WST, and dilution buffer

Thank you for the reference you've sent, I'll be sure to check it out

Hello!

What samples are you using the kit for? What is written in the instructions?

As far as I understand the principle of your kit, the superoxide aninone generated by xanthine oxidase oxidizes the dye. Dye concentration is determined by absorbance at 450 nm. The more superoxide anion is formed per unit time, the more the dye is oxidized and the optical density the faster it decreases over a controlled time. And the higher the activity of superoxide dismutase, the less superoxide anion is formed, which could oxidize the dye. And the higher the SOD activity in your samples, the less the decrease in optical density in a controlled time. Those. in your case, the optical density (450 nm) should decrease. And the smaller the decrease, the higher the SOD activity in the samples you are analyzing. And vice versa.

From my point of view, you have some kind of problem with blank 3 (its optical density should be approximately the same as that of blank 2; both of these blanks do not contain xanthine oxidase - therefore, superoxide anion is not formed in them, which oxidizes the dye). Your blank 3 has a too low optical density (similar to blank 1, which contains xanthine oxidase, but no SOD; therefore, all the superoxide anion that forms interacts with the dye, oxidizing it).

You may have made a mistake when setting the test. Pay attention to blank 3!

The method that I use has a different principle (optical density increases, not decreases; the higher the activity of SOD, the less the increase in optical density at 560 nm).

And how do you do sample preparation?

It is necessary first to homogenize in a suitable buffer, and then centrifuge the resulting homogenate at a more or less high speed (5000-10000 g or more, 10-15 minutes, preferably with cooling). The resulting supernatant should be used for test preparation. It is also desirable to add protease inhibitors to the homogenization buffer.

And I would give you advice to test this kit on serum or plasma.

If you need help, then I am ready to provide you with it!

You can write to me by mail ([email protected]).

Dear colleagues,

All tetrazolium salts (MTT, XTT, WST-1) produce formazan by reduction and accompanied by an increasing OD. In the mentioned kit WST-1 produces formazan and becomes visible (gain OD 450).

>Do I expect the absorbance to increase or decrease with the time?

Increase if the kit works in the right way with your sample.

In the case of a viability study, the source is NAD(P)H-dependent cellular oxidoreductase enzymes. At the same time very often used electron carrier phenazine methosulfate to facilitate the transport of electrons from viable cell structures. Molecules affecting this process could affect the results of the assay a lot. https://en.wikipedia.org/wiki/MTT_assay

> It's not entirely clear why you have such different meanings for blanks?

these blank need only for endpoint assay. In case of dynamic or repetitive measurements (before and after the addition of WST) we do not need these blanks.

>It was an insect tissue, homogenized per instructions in the protocol.

We worked with butterfly pupae extracts in the past and it was very diluted. As well they tend to precipitate, so clarifying by centrifugation before the assay is an obligatory step. I would say you insect extracts could contain too much unidentified molecules that could serve as electron donors or carriers. Simple dilution could shed a light on it.

>Could you please elaborate on the first method a bit further?

Please state which one?

>For example, if absorbance at T0 was 0.250, and five minutes later 0.253, do I note it as +3. What if in the case of some samples it decreased? Do I take it as a negative value then?

If you have a valuable decrease (>5 %) it means something wrong with the assay. It could be cloudy sample dissolving, bubbles, dust in the instrument or even stains/fingerprints/drops on the plate bottom.

And I agree with Aliaksandr - the sample preparation is important as well as testing of the kit using samples with known behavior (a drop of blood could give plasma and erythrocyte lysate as a Positive control).

Daniil

If you have samples you can run in a NATIVE PAGE and some NBT you could try an activity gel. Its not a bad way to compare activities of different samples quickly. It is also possible to run different amounts of an SOD with known activity (U) to make a calibration curve and get approx. units SOD from your samples. Its by no means perfect, and if a comparison is all you need it might be your best way forward.

Dear Lara Ivanković I facing similar issues with a sample from insects, I want to ask you how you consolidate the protocol to compare absorbance. What units did you use (% inhibition or mg protein)