In article <37208D97.D17A73CC at bu.edu>,
julia <julimark at bu.edu> wrote:
> Hi everyone,
>> I'm planning on doing an ELISA next week, but I don't remember (and
> can't find) the protocol that I used before. If you have any tips on
> how to coat a plate and do the assay, I'd really appreciate it!
>> Thanks in advance,
> -Sarah
Hi Sarah,
Coat protein of your interest (5 microgram/ml) in each well of a flat bottom
polystyrene plates in 0.05 M carbonate buffer pH 9.6, containing 0.02% Sodium
azide, with 0.05 ml volume at 37*C for 1h and at 4*C overnight. Block the
unbound sites with 1% bovine serum albumin (BSA) in 0.01 M PBS, pH 7.2
containing 0.05% Tween 20 (BSA-PBS-T) at 37*C for 1h. Add different dilutions
of test samples (0.05 ml) to wells in 1% BSA-PBS-T and incubate the plates
for 1h at 37*C. After addition of horseradish peroxidase (HRP) conjugated
goat anti- mouse IgM or IgG, 0.05 ml/well in 1% BSA-PBS-T, incubate the
plates again for 1h at 37*C. After each step, wash the plates extensively
with PBS-T. Use hydrogen peroxide (0.012%) and 0.04% orthophenylene diamine
(Sigma) in phosphate citrate buffer pH 5.0 in 0.1 ml volume per well as
substrate. After incubation at 28*C for 20 min, arrest the reaction using 2
N Sulfuric acid. Read the optical densities at 490 nm using an ELISA Reader.
Good Luck!!
K.S.Nandakumar
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