Question (37835) | Anti-Catalase antibody (ab52477)

Go to datasheet (ab52477)

Question

Hi,
I had ordered Catalase antibody from Abcam. It worked fine for a few times and then it stooped working. I inquired and the tech support said it might have undergone freeze thaw. So I ordered a new antibody from catalase and made aliquots. It did not work for me. I tried increasing the amount of protein, but it did not work.


Please send me a positive lysate because for some reason I am not able to get results.

Answer

Thank you for contacting us and letting us know about the trouble with this antibody. I am sorry to hear that the results have not been satisfactory.

I looked through our records and found the previous correspondence you had with my colleague about the first vial of ab52477. Could you confirm that the protocol details are the same as before?

1) primary antibody dilution and incubation time - 1:1000 dilution and overnight incubation at 4C.
2) what cell samples used - Used PBT and Jurkat cells
3) how were the samples treated - Samples were lysed and then boiled
4) how much protein loaded - 3ug/sample /well
5) gel electrophoresis conditions - 125 Volts for 90 mins and transferred at 25 volts for 90 mins
6) transfer and blocking conditions (how long and what reagent was used) - 3% BSA for blocking, Primary and Secondary.
7) secondary antibody dilution and incubation time - HRP, 1:2000 dilution and 1 hr incubation. Western using other antibodies with the same secondary gave me nice results.
8) detection method used - super signal west pico chemiluminescent substrate

I also have a couple of additional questions. What was the largest amount of protein that was loaded per lane of the gel?Was the protein transfer verifiedwith a Ponceau stain?

We do guarantee all of our products to work as stated on the datasheets for up to 6 months after purchase.I would consider Jurkat cells and PBTs to be positive controls, as I would expect them to beexpressingcatalase, so I would be happy to send you a different antibody if you would like. We also have ab1877, which has been testedwith human cells in Western blot-

https://www.abcam.com/Catalase-antibody-Peroxisome-Marker-ab1877.html

I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that we can do for you.

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