SOD assay

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angelaeds
angelaeds's picture
SOD assay

Hi everyone,
i am in a very perplexed position. i have carried out the SOD assay using the NBT method. if im not wrong, the control solution (without plant sample) should give the darkest colour after 15 minutes of reaction time, while hte rest of the plant samples should be lighter, assuming the samples had some amount of SOD enzyme.
but instead, 2 of my plant samples became darker than the control solution. please help, i do not know what to do.

thanks

R Bishop
R Bishop's picture
angelaeds,

angelaeds,

I have a couple of questions to get started helping you out. 

First, are all your plant samples extracted in the same buffer and the same volume and same mass of plant material?

What buffer are they extracted in?

Are you using a kit to perform the SOD assay or making your own reagents?

How did your standard curve look? Was it linear in triplicate assay conditions?

Next did you try serial dilutions of the plant material or just one concentration.  Often times this is a step people miss and can lead to results like yours.

We can work from there. My guess is the plant material mass is too hi, bringing in contaiminants that are affecting the NBT emission, but no way to be sure of that.

Bishop

angelaeds
angelaeds's picture
Plant tissues (0.5-1.0 g) as

Plant tissues (0.5-1.0 g) as homogenized with 100 mMpotassium phosphate (pH 7.5) containing 2 mM EDTA and % PVP-40 at 4deg C. The homogenate was filtered through four layers of cheesecloth and centrifuged at 15,000g for 20min. Protein content in the supernatant was concentrated by ammonium sulfate precipitation. Ammonium sulfate fractions between 45 and 90% were pooled and extensively dialyzed against the same buffer with three changes of buffer overnight at 4°C. Dialyzed and concentrated protein extracts were stored at -80°C for further analysis (Rao 1996). The activities of different enzymes were determined with plant extracts equivalent to 100 µg of protein. Protein content is determined according to Bradford (1976) with BSA as standard.
Im not using a kit, preparing buffers on my own. Therefore, there is no standard curve to produce.
No i did not try serial dilutions of the plant material.
Could my mistake be not adding the xanthine oxidase to generate the superoxide? could that be the problem? I suppose general descriptions of the method do not state that as it may be assumed i use that

R Bishop
R Bishop's picture
That is likely the problem. 

That is likely the problem.  You are not actually generating superoxide at all without the xanthine oxidase.

SOD assay reagents

50mM Phosphate Buffer pH 7.8
0.1 mM EDTA
0.01 mM ferricytochrome C
0.05 mM xanthine
6nM xanthine oxidase

total of 3mls from my notes.  I got this protocol out of the book Mitochondria, which is now available from Google books for free.

You can buy SOD from Sigma to run a standard curve and insure your assay is running correctly. In fact, before you thaw another sample,I would several test runs and get that standard curve looking like a champion.  Your results will be a lot better and the most important thing is you can quantitate the SOD activity in your samples.

Best wishes

Bishop

angelaeds
angelaeds's picture
Thank you for the protocol. I

Thank you for the protocol. I will try that. 

However, over here in Malaysia, chemicals are not 'shelf ready', but haf to be ordered and this will take some time.

Therefore, after going through an article using similar method as i do;
Giannopolitis and Ries, 1977. Superoxide dismutases. Plant Physiol 59: 309-314

i realise that they use flourescent light to generate the superoxides, which i did apply to my method as stated below:
 
Total SOD activity was determined by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium chloride (NBT) as described by Giannopolitis and Ries (1977).
NBT reaction with O2- would generate water-insoluble blue formazan dye.
The reaction mixture (1.5 mL) contained 50mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13mM methionine, 75 mM NBT, 2 mM riboflavin and 50 mL enzyme extract.

Riboflavin (to homogenize formazan) was added last and tubes were shaken and illuminated with a two 20-W fluorescent tubes.

The reaction would be allowed to proceed for 15 min after which the lights would be switched off and the tubes covered with a black cloth. Absorbance of the reaction mixture would be read at 560 nm.

One unit of SOD activity would be defined as the amount of enzyme required to cause 50% inhibition of the NBT photoreduction rate and the results expressed as Umg-1 of dry mass (DM).

Please can may you give me a feed back on this method? Thank you

PedroS
PedroS's picture
Hello everyone,

Hello everyone,

Talking about SOD, I'm trying to measure it using Xanthine Oxidase/NBT assay but something is wrong I after I read these posts I hope someone could help me... so here it goes...

My Reagents:
Buffer - 50 mM Sodium Phosphate Buffer + 3 mM EDTA (solution's pH 8.0)
Xanthine's Stock Solution prepared in NaOH 1M and diluted to 7,5 mM in buffer
0,75 mM NBT prepared in buffer
0,06 U/mL Xanthine Oxidase (XO)
SOD is prepared in buffer.

I'm measuring the activity of commercial bovine SOD (sigma) and during all my experiments strange things happen:
1. when I put together XO, NBT and Xanthine I don't observe an increase in Absorvance as it was expected with the reduction of NBT to formazan (solution doesn't turn blue).
2. when I put the whole system together (XO, Xanthine, NBT and SOD) the solution turns blue and an increase in absorvance is observed.
3. An increase in absorvance is also observed when I put together NBT, Xanthine and SOD (without XO) but solution with NBT and Xanthine alone or together don't turn blue (only happens when I add SOD)...

So... Xanthine Oxidase doesn't seem to work... SOD seems to reduce NBT in the presence or absence of Xanthine Oxidase....  Is there any undesired source of reducing agents?

Can anyone help me?
Pedro

cassavagirl
cassavagirl's picture
Hi angelaeds,

Hi angelaeds,

I am new to enzyme assay. Now trying exactly the same method you used but  with microplate. I tend to get a very low abs for reaction without enzyme ~0.05 compared to those containing extracts which >0.6. Is this what I suppose to get?

Also, can you please show me how to calculate the SOD activity?

Thank you

mahigene
mahigene's picture
hello everyone,

hello everyone,

angelaeds,

that is the right abs what u have, i  also use Giannopolitis and Ries, 1977 method and find the abs much fewer what u mentioned near about .048. and useing this method i did experiment near about 40 times

sobasco
sobasco's picture
hi every one,

hi every one,
i need little guidance regarding the protocols about SOD & CAT assay & choline both  by using  fresh and dry plant materials.
proper method to find it

thanks

Lusanda Matee Matee
Lusanda Matee Matee's picture
SOD assay problem

Hi everyone, I've been trying the SOD assay on lichen material using the Beyer and Fridovich method and I'm getting higher values for the samples than the control without the enzyme extracts , these values are just the absorbtion values or OD values. I have not done an inhibition curve yet to calculate the actual value ,these values are juist as they are. Am I doing the right thing and will the values be higher once I've used the 50% inhibition formula ?