mdp96tab at my-deja.com
Wed Feb 23 12:52:38 EST 2000
In article <87l6cc$jgq$1 at nnrp1.deja.com>,
jack_horner at my-deja.com wrote:
> From the method I use:
> 1. Antigen (peptide/protein) was diluted to 5 mg/ml in
> carbonate-bicarbonate buffer (see MATERIALS below). For example, 5 ml of
> peptide from a 10 mg/ml peptide stock was added to 10 mls of
> carbonate-bicarbonate buffer (enough for one plate). 100 ml per well was
> added to flat bottomed polyvinyl chloride microplates (Flow Laboratories
> Inc.) and incubated overnight at 4oC or 90 minutes at 37oC.
> 2. Antigen was flicked off the plate and the wells were blocked with 200
> ml of 5% skim milk in PBS-Tween 20 (see MATERIALS below) overnight at
> 4oC or 90 mins at 37oC.
> Carbonate-bicarbonate coating buffer
> 15 mM Na2CO3 (1.6 g)
> 35 mM NaHCO3 (2.9 g)
> 1 litre H2O
> The pH was adjusted to 9.5
> And now a question: wouldn't a method that used drying of Ag to the
> plate result in free antigen (that would competitively bind antibodies)
> being released when the test serum was added? Or do you wash it off
> using the blocking agent.
> martinC at qimr.edu.au
Sorry for the delay in replying. I presume drying antigen onto a
plate would result in free antigen. You assume correctly...free antigen
is washed from the plate before blocking. I guess it's just a desperate
attempt to get peptide to stick down ( and beleive me I was desperate!).
Thanks to all for your advice
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