ELISA- triton in coating buffer - unconventional plate coating (Nov/16/2005 )
Dear all,
I am a real beginner in ELISA. I intend to coat my ELISA plates with a protein extract containing 0.5-1% triton X-100. I know it is not very conventional.
Has anybody got any experience with different plate coating buffers (i.e. detergent in buffer)? Do I need a different ELISA plate for that?
All the best!
Diril
I am a real beginner in ELISA. I intend to coat my ELISA plates with a protein extract containing 0.5-1% triton X-100. I know it is not very conventional.
Has anybody got any experience with different plate coating buffers (i.e. detergent in buffer)? Do I need a different ELISA plate for that?
All the best!
Diril
hi,
in my view detergent is not necessary while prtein coating in ELISA. that too triton x-100 is a strong detergent, which may damage your protein(i never used triton x in my ELISA) or may bring strange results.
you can coat protein with 1xPBS or 50mM tris+ 150mMNaCl or coating buffer (bicarbonate buffer.pH-9.6).
when you use detergent in ELISA, it will reduce nonspecific binding. 0.05%tween20 universally accepted concentration in primary, secondary Ab solutions and in wash buffer.
regarding ELISA plate it is better to use, high binding(maxisorbant) plates from Nunc.
i hope above information will help you
gud luk for your research
sravan k.payeli
I am a real beginner in ELISA. I intend to coat my ELISA plates with a protein extract containing 0.5-1% triton X-100. I know it is not very conventional.
Has anybody got any experience with different plate coating buffers (i.e. detergent in buffer)? Do I need a different ELISA plate for that?
All the best!
Diril
Do you need to dilute your protein for coating ? If so the just dilute it in pBS cause Triton like Tween is rather used to wash unbound proteins or blocking free sites.
If you intend to use your protein very concentrated youcan also dialyse it against pBS or TBS depending which plate you plan to use !
pesji
Thank you both for your advice. I guess I will need to get rid of most of the triton in my protein extract either by dialysis or by extensive dilution. Otherwise the proteins won't stick to the plates, right?
Well I hope it works!
Best wishes,
Diril 
Hi,
I use an extract containing Triton as well. It works fine for my Western blots so I don't see any reason that it won't work for ELISA's as well. What sort of ELISA are you performing? I don't think that the Triton should cause significant problems.
I use an extract containing Triton as well. It works fine for my Western blots so I don't see any reason that it won't work for ELISA's as well. What sort of ELISA are you performing? I don't think that the Triton should cause significant problems.
Thanks for the message.
Hmm, the ELISA I want to perform is aimed at the detection of an internalized antibody. Ideally some cell types should internalize this antibody and others shall not. Then I want to lyse the cells with triton and coat the ELISA wells with this lysate. Next I will add an enzyme conjugated 2dary to detect the internalized primary ab.
I thought I may have problems with coating the plate with presence of triton in the extract.
Soon I do a test dilution in the presence or absence of triton to check.
Regards,