I have used the following protocol to asses the catalase activity in the homogenized mouse stomach samples infected with H.Pylori.
In the Pre-test we have used 8 standard samples with different dilutions with catalase standard dilution and standard buffer and we have included three other Normal control samples.
However,the Spectrophotometry reading for standards gives almost the same results for all samples.
Although we have repeated the same test for several times and the results were the same( no any visible color gradient )
What are the Suggestions and recommendations for this issue?
If you mean testing tissue homogenate for catalase activity, the test is not reliable because red blood cells, which are unavoidably present in the biopsy, possess a strong catalase activity that would mask and overwhelm the H. pylori catalase.
Dear professor Natale Figura,
Thank you very much for your valuable answer.
In our case,We usually add RIPA buffer and protease tablets to the initial sample and then the Homogenization step.
Would this step be useful in eliminating the catalase activity of red blood cells?
The red blood cells, present in the biopsy, possess a catalase activity that would mask H. pylori catalase.
If you sue a protease to clean your sample the protease activity will affect H. pylori catalase. Also if you do not have visible color gradient it is because your enzymatic assay is saturated
Dear professor Héctor Toledo
Thank you very much for your answer
Would you please explain why we did not get a color gradient for the standards.?
Dear professor Héctor Toledo
Please find the attached protocol for catalase test which we've been using in our lab.
Your valuable suggestions will be highly appreciated
Dr Toledo is right and seems to know the subject better than I do. I quit
To make the standard curve the first thing it is to prepare a catalase standard solution at 10,000 Unit/mL from the catalase standard stock (600,000 units/mL). So you will have your first tube, # 1, to start the standard curve. From this tube you have to do a serial dilution (1:2) one by one until tube 7. If you follow that protocol the curve in the tube 1 will have 100 units/mL and the tube 7, 1.56 units/mL. Other important step in the assay protocol is the step 3 to stop the reaction if not the catalase will continue working. If you follow each step you should have your standard solution. I don´t have other advise. I hope you could find this OK and the next time you build a good standard curve.
Thanks Dr. Figura, your advice was OK and also after this I quit
Dear Professor Héctor Toledo and Natale Figura
I immensely appreciate your valuable suggestions on my issue.
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