I'm having the problem that in my SOD assay (NBT assay, Pyrogallol assay) the sample reaction is showing a faster O2- production than the blank of the SOD standards. Means that the samples are way darker in color after 5min of reaction than the SOD blank. I'm working with Sephadex desalted pine crude extracts.
Do you have a literature reference for the SOD assay you use? If you use a spectrophotometric assay there could be a high level of interference by some compounds in the pine crude extract. In this case you could use a native gel assay for the determination of SOD activity. Here you will find a good paper on this method: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2830880/
Cheers, Christian
Correct me if I am wrong but I believe NBT stimulates the production of superoxide (O2-) which is dismutated to O2 or H2O2 by SOD. Considering this, the SOD standards should be BLANKS, this is, no or little superoxide (O2-) should be detectable in comparison to your samples in which O2- production may vary.
To summarize, samples ARE supposed to be darker than SOD blanks, at least the assays says else. Check it.
@ Luis No in the SOD standard curve SOD dismutates O2- and stops the colour reaction. So the blank SOD standard has the darkest colour.. But the samples are darker.. @ Christian i use http://www.ncbi.nlm.nih.gov/pubmed/22656066 for Pyrogallol assay and http://www.ncbi.nlm.nih.gov/pubmed/21282325 for NBT assay.. Both show the same phenomenon. I also think of some interfering compound but i though Sephadex desalting and PVPP in the extraction of proteins stop that. With crude extract i mean that i only do a aqueous protein extraction with phospahte buffer,PVPP and Triton X-100.. Thanks for the answerd so far
My explanation isn't wrong then, it is just that I misundertood the concept of "SOD blank". Anyway, have you tried running the extract without Sephadex and PVPP purifications? That is for the samples. As for the assay, I can only think of a contamination of the blank. SOD is an enzyme and enzymes are highly labile, cross contaminations aren't unusual. My recomendation to avoid contaminations is to handle SOD blank before you handle the rest of the standards.
The best of luck.
@ luis thanks for the quick reply.. I had the problems without sephadex and without PVPP as well, i thought this would solve it by catching phenolics with PVPP and Sephadex desalting to get rid of low molecular stuff.. And nbt is not stimulating the O2- production it is the phenazine methosulfate (PMS) that does it.. For the SOD standards. I do a dilution series and they work well. So the assay is okay it is something in the extracts..
You could try Amicon columns (from Merck Millipore) for desalting (or changing the buffer). They are quite efficient at this task.
Regards, Christian
Yes. If the amount of peroxide is beyond the capacity of dismutases in the kidnapping of superoxide anion, all the antioxidant system can go into imbalance and or cytotoxicity and thus immobilize the activity of superoxide dismutase. See my article Sleep deprivation alters gene expression and antioxidant enzyme activity in mice splenocytes and quotes Feissner and Atli in the references of this article
Thanks Lisandro Lungato for the answer.. I found a paper from 1989 from Polle et. al. with spruce extracts prepared the same way as my pine samples were catalase is in the assay buffer of the SOD activity test http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1061888/pdf/plntphys00643-0088.pdf . I'll give this assay a try and will also add Catalase for a test in the pyrogallol or NBT assay. This might solve the problem... Many thanks
no they are different in level and causes of level changes, its propably due to extract other compounds interferences
So far none of the assays worked unfortunately... But thanks for your hints