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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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triton is affecting protein detection
Posted by: wender (IP Hidden, New member, 2)
Date: December 5, 2005 07:29AM
Hi,
For some strange reason when I add triton to cell lysates my protein is no longer detected on a western blot. Does anyone have any idea as to what could be causing this? Can I learn something from this about my protein? Any advice will be a great help. Thanks! w
Re: triton is affecting protein detection
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: December 5, 2005 09:08AM
It may indicate: 1. cell lysate buffer is much weaker and can no longer break membranes and release soluble proteins. OR 2. proteins are degraded for some reason due to adding of triton.
Triton series are commonly used non-ionic mild detergent for protein extraction and should be suitable for most of protein extraction. If you consistantly have this problem even after changing batch of triton, you may want to try other detergents and do a comparison. But I doubt the triton has anything to do with the characteristics of the protein of your interest.
Re: triton is affecting protein detection
Posted by: wender (IP Hidden, New member, 2)
Date: December 5, 2005 11:23AM
Thanks for you reply!
"It may indicate: 1. cell lysate buffer is much weaker and can no longer break membranes and release soluble proteins. OR 2. proteins are degraded for some reason due to adding of triton." Regarding your suggestions: 1)My protein is not detected in any fraction including total lysate and membrane fraction. Therefore, even if the buffer is weak as you suggested, the proteins should still be in one of these fractions in its insoluble form. 2) Do you have any idea if triton can cause proteins to get degraded? or to not be detected any longer by their antibody? Thanks again!
Re: triton is affecting protein detection
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: December 7, 2005 09:26AM
wender Wrote:
------------------------------------------------------- > 1)My protein is not detected in any fraction > including total lysate and membrane fraction. > Therefore, even if the buffer is weak as you > suggested, the proteins should still be in one of > these fractions in its insoluble form. Then I would suspect whether the western is working or not, particularly the antibodies. Did you perform control using other Ab? > 2) Do you have any idea if triton can cause > proteins to get degraded? or to not be detected > any longer by their antibody? No. I have never heard that triton has anything to do with protein degradation. Best luck.
Re: triton is affecting protein detection
Posted by: shahid (IP Hidden, New member, 2)
Date: February 19, 2006 10:14PM
dear! Triton x-100 is a stabilizer for some proteins like cholesterol ester hydrolase. we use it in extraction buffer.
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