Kindly help me to choose the appropriate method for estimation of Cox II in diabetic rats, ELISA or western blotting.
Hi...
I would suggest to check expression as well as activity both.
As Alexis Broquet mentioned, you can use PCR and Western blot to check expression levels. Expression at protein level using Western blot should be sufficient.
Enzyme assay kit would be sufficient to measure activity.
Both expression as well as activity are important.
What about an enzyme assay that is infinitely easier and quicker. I just looked on the internet. There are several kits commercially available.
The kit states that The cyclooxygenase component converts arachidonic acid to a hydroperoxy endoperoxide (Prostaglandin G2; PGG2), and the peroxidase component reduces the endoperoxide to the corresponding alcohol (prostaglandin H2; PGH2), the precursor of prostaglandins, thromboxanes, and prostacyclins.
They state that the peroxidase activity is monitored by the appearance of oxidized N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) at 590 nm.4 It can be used with both crude (cell lysates/tissue homogenates) and purified enzyme preparations. The kit includes isozyme-specific inhibitors for distinguishing COX-2 activity from COX-1 activity.
Hi,
What do you want to estimate? Cox-2 expression and/or activity?
Depending on your choice, the answer is different.
for expression: qPCR or western blot
for activity: an ELISA kit or enzyme assay as said by Marcia Moss.
alexis
Hi...
I would suggest to check expression as well as activity both.
As Alexis Broquet mentioned, you can use PCR and Western blot to check expression levels. Expression at protein level using Western blot should be sufficient.
Enzyme assay kit would be sufficient to measure activity.
Both expression as well as activity are important.
Depending on what you are looking for (expression or activity) I would even consider immunohistochemistry. However, above suggested ideas are great, l buy them. Good luck.
Hi Devendra,
as the comments suggested above by Chintan, Alexis and Marcia is pretty much the same what I was going to suggest.
Real time PCR (qRT-PCR) should give you the gene expression results at the molecular level, however as you can't conclude your results just by gene expression studies, I would run western blot to measure if the gene has translated into a protein and also an ELISA to know the functional endpoint of the enzyme in the clinical condition you are looking in rats.
Good luck
Sesh
Western blotting is more accurate than ELISA, but it is much expensive and Time-consuming
On the contrary Ayat, ELISA assay is a quantitative whereas WB is at best semi quantitative. So if possible ELISA is to prefer. However, I agree with Marcia, when it comes to enzymes their biological activity is most important. You hence may gain a better or more relevant to your objectives than its protein level or gene expression!
I am totally agree with Taher. ELISA is preferable compared to WB in terms of accurate quantification. But as WB is much cheaper compared to ELISA, it can be used along with activity assay.
I disagree with Ranu. ELISA cannot measure activity of an enzyme. It can be used only for quantification. For activity measurements there are enzyme activity kits available. However, in an alternative approach, traditional enzyme activity assays can be used which are much cheaper than kits.
I would suggest to read the article (ref: Clinical Infectious Diseases 2001;32:1114–1114) to understand basic differences between ELISA and Western blot and that might help you to decide what and which test to use in your studies.
Dear Devendra,
which kind of samples do you investigate? ELISAs for relevant prostaglandins (COX-2 products) are applicable to body fluids and cell/tissue culture supernatants. Western blotting is a good choice for (semi)quantification of protein levels in cells and tissues. Immunohistochemical staining of tissues provides information about the cellular localisation of proteins and is a rough estimate for expression levels. Determination of COX-2 mRNA in cell culture and tissues provides data on gene gene expression which does not necessarily correlate to protein expression. Activities of intracellular enzymes are usually measured with purified enzymes or in homogenates.
What do you want to estimate? Cox-2 expression or activity?
The answer is different.
for expression: PCR or western blot
for activity: an ELISA kit or enzyme assay as said by Marcia Moss and Alexis Broquet
Dear Devendra,
I agree with all above comments and suggestions, But I think it mainly depend on how many samples you have? and what is your budget? as ELISA is more expensive when u compare to WB and qRT-PCR.
Hello Devendra, I think it will be appropriate if you employ both WB and ELISA. You can first use WB to verify whether the expression was successful since it is relatively cheaper to perform compared to ELISA. If your WB prove positive, you can then use ElISA to verify its (your protein) activity. All the best.
I suggest to use Western blot analysis which is more accurate than ELISA.
Have a look on this paper:
Insulin Reduces Cerebral Ischemia/Reperfusion Injury in the Hippocampus of Diabetic Rats
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