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ELISA background problem - (Dec/07/2007 )

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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

-biojwb-

non specific binding

try blocking in 1-3 % BSA (10-30 mg/mL)

maybe coating problem
how much peptide do you coat?
use maxisorb plates
try different coating buffers like carbonate buffer and phosphate buffer

-Missele-

QUOTE (Missele @ Dec 7 2007, 12:14 PM)
non specific binding

try blocking in 1-3 % BSA (10-30 mg/mL)

maybe coating problem
how much peptide do you coat?
use maxisorb plates
try different coating buffers like carbonate buffer and phosphate buffer



i coat 10 ug/ml target
and use 1ug/ml for peptides

-biojwb-

i also use maxisorp plates

-biojwb-

probably the coating problem. try to coat it overnight at 4C

-Sandwich-

do you think the peptide molecules are so small that they have steoric hindrance to access Ab after blocking with the big BSA (ca. 60kD)?

-Sandwich-

It could be that the BSA is too big as the peptides are only 7mers and the target is only a 12 mer. I did try using nickel chelate plates to adress this problem but with the same results. It seems to me that the streptavidin is binding to everything as i still get colour in control wells which contain the coating target. Also i do usually coat overnight

-biojwb-

QUOTE (Sandwich @ Dec 10 2007, 07:40 PM)
do you think the peptide molecules are so small that they have steoric hindrance to access Ab after blocking with the big BSA (ca. 60kD)?

It could be that the BSA is too big as the peptides are only 7mers and the target is only a 12 mer. I did try using nickel chelate plates to adress this problem but with the same results. It seems to me that the streptavidin is binding to everything as i still get colour in control wells which contain the coating target. Also i do usually coat overnight

-biojwb-

Hi,
what is the sequence of the peptide you are using to coat
may be try using sodium bicarbonate buffer pH 9.6 where coating is very good and maxisorp plates is the best option
all the best

-leelaram-

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

-Ceri-

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