I am looking for laboratory biochemical assays for tissue samples to estimate Catalase, Glutathione Peroxidase, Superoxide Dismutase in an animal tissue sample.
Alternative to Gel Activity method.
I am not able to find this protocol online. Could someone help me out on this?
Dear Lokesh
please find this document. since i have done the biochemical assays using serum samples you still can follow them for the tissue homegenate.
If you need anything please let me know.
i would be very happy to help you out
i agree with Shreewardhan methods, as we follow same protocols with minor changes.
Thank you very much for your reply Shreewardhan.
I find that attachment very useful. I will definitely try all the methods.
I m sure, many others will be benefited from this. Very helpful and useful document.
Thanks Again
LoKesH
Dear Lokesh
thanks for replying..
i have done the same assays in my lab and got the good results out of it.
best luck for your work
Dear Shreewardhan, May I know what is the percentage of homogenate you suggest? Which buffer is best?
Better you use KPB pH 7.4 for homogenate, Better you perfuse your tissues with 1.15% KCl and then Homogenate with KPB, If You are taking 1gm of tissue then use 4ml of buffer vice versa.
hope this works out for you.. good luck
I am working with brain tissue and I use PBS, pH 7.4 and make a 10% homogenate. Can I directly use this homogenate (4-5 mg/ml protein) for biochemical assays? I use 50-100ul of sample or is more dilution needed?
No, you can use homogenate for lipid peroxidation and GSH estimation whereas for enzyme kinetics like catalase, SOD, GPx and GR better use supernatant. Take the homogenate centrifuge it at 15000rpm at 4C for 1hr and collect the supernatant, make aliquots for each assay as you can avoid freeze thaw cycle. I think you can use brain sample directly whereras in case of liver, protein content will be more. if you think protein conc is more, take less sample for the assay.
i think so if sampling volume is problem then better to follow ELISA method where you can reduce one tenth of sample in place of normal manual method because in ELISA method just we have to use sample in microlitre while in normal method have to used in ml.
Dear Arbind, ELISA method is too expensive whereas this are regularly used protocols, where you hav to standardize the experiment by changing substrate conc or protein conc or pH whatever it may be. EXperiment does not depend on microlitre or ml it depends on the protein conc it is having. Did you see anybody using ELISA for enzyme kinetics??? I dont think soo.
Dear Arbind i agree since you can use the newer methods for the detection but ELISAs are costly things .again there are some companies which have colorimetrically based ready to use kits but again the cost is too high. whereas making the lab chemical based thing is much cheaper and good results can be obtained.
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