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How to optimise Western blotting for eNOS detection?

How to optimise Western blotting for eNOS detection? - Molecular Biology Techniques

How to optimise Western blotting for eNOS detection? - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 02-12-2010, 02:30 PM
Pipette Filler
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Default How to optimise Western blotting for eNOS detection?



I am working with Westren blotting to find the expression of eNOS, other colleagues from my department told me that eNOS is a very difficult protein to be detected. How can I manage this? On my membrane, the same sample with same concentration of the positive control (lung and kidney of mice) applied on different gels has shown bands with GAPDH, caveolin and COX-2, but no band on the gel to find eNOS. Can you help me to explain why? and how to optimise my result? My primary antibody was Polyclonal Rabbit Anti-eNOS/NOS Type III from BD Transduction. Both of the primary (1:1000) and secondary antibodies (1:1000) are dilute in skim milk 5% (TBS-Tween 20).
PS: I also work with cell culture (BAEC), in my 1st experiment to lysate the cells with RIPA, I can detect eNOS easily; however with the second time, I add orthovanadate into RIPA, I could not find the band for eNOS in Western blotting? I tried to find in literature, but orthovanadate could not be the cause for the problem. Would you please give me some comments? Thank you very much!
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Old 05-04-2010, 02:23 PM
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Default Re: How to optimise Western blotting for eNOS detection?

Hi,
try do dilute your milk to 1% while incubating your primary and 0.5% for your secondary ! It works for us... How much proteins do you use ?
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Old 10-11-2010, 09:42 AM
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Default Re: How to optimise Western blotting for eNOS detection?

Are you certain you have the right antibody? eNOS is type II NOS, not III - Thats iNOS.
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Old 10-18-2010, 04:14 AM
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Default Re: How to optimise Western blotting for eNOS detection?

@ gregmey: thank you, I will try. Recently, I heard from one of my colleague, she was successful with finding eNOS, but she used heart tissue and eNOS antibody fom ABCAM (not from BD transduction) as me. Is one of the reason?
Moreover, I have one more question:
- after the electrophoresis, and tranfering to the membrane, I cut the membrane into parts
- The upper part, I did with eNOS antibody

- The lower part, I did with COX-2 antibody

- However, I do not know why I always have a lot of background with eNOS? although I did the same method.
NOTE: eNOS is my first antibody (the membrane has never had any contact with any other antibodies before eNOS)
I made a varriation from 1-2 hours blocking the membrane with skim milk.
@ Gibberling: yes, I used the right eNOS, the name type III is written on the information document of the manufacture.
Attached Images
File Type: jpg eNOS.jpg (9.4 KB, 11 views)
File Type: jpg Q3min-cox2.jpg (5.5 KB, 6 views)
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Old 04-17-2013, 02:11 PM
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Default Re: How to optimise Western blotting for eNOS detection?

I have the same issue in detecting eNOS in lung tissue (no clear signal). I diluted my primary antibody in a 5% milk solution and will try with the 1% solution. The antibody is from SantaCruz. Moreover, I would like to find eNOS uncoupling. Any other experiences and possible tricks are more than welcome. Thanks!
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blotting , detection , enos , optimise , western


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