PBS (Phosphate-Buffered Saline, Ca/Mg free) :-
N.B.: Na2HPO4.2H2O can also be used -> add 1.42g for 1x and 14.2g for 10x PBS.
Make up to 1 litre with dd H2O.
100ml 1x PBS
10ul Tween (0.01%, final concentration)
Citrate/Acetate Buffer pH6.0:-
Sodium acetate - 4.1g.
dd H2O - 500ml.
2. 100ml 0.1M Citric acid
Citric acid - 2.1g.
dd H2O - 100ml.
Titrate 1 with 2 to a final pH of 6.0. Citrate/Acetate Buffer, pH6 can be stored frozen at -20°C.
TMB Solution - 10mg/ml in DMSO (store at 4°C).
The following details the method for use with TMB as the substrate (cover wells with clingfilm during incubations to prevent evaporation).
1) Add 3ul H2O2 to 20ml of Citrate/Acetate buffer. To every 5ml of buffer add 50ul TMB solution.
N.B.: Colour change is from colourless to blue (yellow when acid is added).
2) Coat plate with antigen stock solution (1ug/ml in PBS, 50ul/well) for at least 24hrs or over the weekend at 4°C or O/N at 37°C.
3) Remove antigen and wash 3x with 1x PBS.
4) Block with 1% BSA (100ul/well) in 1x PBS for at least 1hr at 37°C.
5) Remove BSA by flicking plate.
6) Wash 3x with 1x PBS containing 0.01% tween
7) Add anti-serum (diluted as appropiate, 1/100 etc. with 1x PBS) and incubate for 2hrs @ 37°C.
N.B.: Use positive and negative controls.
8) Wash 4x with 1x PBS containing 0.01% tween.
9) Add peroxidase conjugate (50ul/well) diluted 1:1000 with 1% BSA (diluted in washing solution as in 8)) and incubate for 1hr @ 37°C.
N.B.: Use PRAM (Peroxidase-conjugated Rabbit Anti-Mouse) for Ab raised in mice and PSAR (Peroxidase-conjugated Swine Anti Rabbit) for Ab raised in rabbits.
10) Wash 4x with 1x PBS containing 0.01% tween.
11) Add substrate (100ul/well).
12) Incubate for 1-5 mins in the dark at RT. Check reaction for colour change. Wells should turn blue.
13) Stop reaction by the addition of 25ul 0.1M H2SO4/well.
14) Read OD @ 450nm for TMB.
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Click here to return to the Submission Form or here to go to the Methods Finder.