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ELISA background problem - (Dec/07/2007 )

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QUOTE (Ceri @ Dec 12 2007, 01:14 PM)
I'd agree with the other posts from Missele and Leelaram try a carbonate/phosphate buffer to coat or maybe PBS. Plus coat overnight at 4C or room temp.

I also wanted to ask about the buffer you incubate in 0.1M NaHCO3 pH8.6. Is this buffer/pH important for the binding? As you then use PBS-T or TBS-T for the wash steps. If it's not important why not dilute your peptides in 1% BSA/PBS and your Strept-HRP step in 1% BSA/PBS.

Our strep-HRP dilution is 1:200 (R&D) or 1:1000 (Amersham) but I guess that will depend on the manufacturer of your strep-HRP. I don't think you need to change to TBS-T for the last wash we don't do this for any of our ELISAs, just use PBS-T all the way through (3x 250┬Ál/well by hand or with a plate washer for each wash step) .

If you're not getting any effect over background maybe you need to increase the concentrations of both that you use or maybe try coating the plate with strepavidin and bind the biotinylated peptides to this and then add the other peptide and detect with an anti-His tag antibody. (I'm assuming your target is His tagged as you mentioned using Nickel coated plates).

Hope this helps in some way.

Ceri

Hi thanks for your advice. Firstly i dont think th ph is important for binding. i will try diluting in PBST+BSA. and STREP in pbs+bsa. I have also tried coating the plates with streptavidin as you suggest but unfortunatly that did not seem to work either. i am starting to think that the peptides do not bind?

-biojwb-

Ceri[/quote]
Hi thanks for your advice. Firstly i dont think th ph is important for binding. i will try diluting in PBST+BSA. and STREP in pbs+bsa. I have also tried coating the plates with streptavidin as you suggest but unfortunatly that did not seem to work either. i am starting to think that the peptides do not bind?
[/quote]

We don't use tween 20 in the incubation steps just in the washes.

Ceri

-Ceri-

Dear all,

I am a student starting to do ELISA. I just cannot get rid of the background signal. Need some expert advice.
I coat 96 well plate overnight with 12ug of streptavidin in PBS (I want to saturate the well)
Then, I washed 3 times with TBST
Block with 5% Milk in PBS for 1 hour
Add 20ug of Biotin-HRP in 5% milk for 1 hour
Wash 3 times
100ul of TMB
100ul of H2SO4 to stop the reaction
Read absorbance at 450nm

When I re-do this protocol on empty wells (no strep), I have super high signal (comparable to streptavidin coated wells).
Are there any problems with the protocol? I had also tried BSA
Help.. Thanks guys!

-studentL-

QUOTE (studentL @ Dec 2 2008, 02:59 AM)
Dear all,

I am a student starting to do ELISA. I just cannot get rid of the background signal. Need some expert advice.
I coat 96 well plate overnight with 12ug of streptavidin in PBS (I want to saturate the well)
Then, I washed 3 times with TBST
Block with 5% Milk in PBS for 1 hour
Add 20ug of Biotin-HRP in 5% milk for 1 hour
Wash 3 times
100ul of TMB
100ul of H2SO4 to stop the reaction
Read absorbance at 450nm

When I re-do this protocol on empty wells (no strep), I have super high signal (comparable to streptavidin coated wells).
Are there any problems with the protocol? I had also tried BSA
Help.. Thanks guys!


may try sodium bicarbonate buffer pH 9.6 for coating buffer instead of PBS

I found out that using sodium bicarbonate reduce the background reading.

-Minnie Mouse-

QUOTE (biojwb @ Dec 7 2007, 11:57 AM)
Hi i need a elisa expert. i am having trouble with an elisa protocol. Background to the problem:. I have used phage display to identify peptides that attach to a target. I then isolated and sequenced these peptides and have had them synthesised. Now, i need to verify by using Elisa that these peptide sequences do bind to the target as they did during page display. here is my elisa protocol;
Target suspended in 10mMNa borate and plates coated 2h/30c
Washed pbst
Blocked , 0.1M NaHCO3 pH8.6 +5mg/ml BSA
Washed PBST
Synthesised peptides (biotinylated) in 0.1M NaHCO3 pH8.6 +5mg/ml BSA diluted down the plate with a staring conc 1ug/ml. 2h/30c
Washed PBST
1:5000 HRP-streptavidin diluted in PBST and added to plates 1h/30c
Washed TBST
100ul TMB substrate added watch for colour change.

When i choose one target i do not see much colour change i.e no binding?
if i use a second target that i also raised peptides against i get a colour change but the controls also are the same colour. non specific binding?

Please help


How do you do the biotinylation, and which aa's do your peptides contain? The biotin molecules can prevent target recognition of the peptides - after all you modify them, and thus alter their shape.

-SatanClaus-

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