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Bicarbonate/carbonate coating buffer (100 mM)
Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:
3.03 g Na2CO3
6.0 g NaHCO3
1000 ml distilled water
1.16 g Na2HPO4,
0.1 g KCl
0.1 g K3PO4,
4.0 g NaCl (500 ml distilled water) pH 7.4.
Commonly used blocking agents are 1% BSA , serum, non-fat dry milk, casein, gelatin in PBS.
Usually PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).
Antibody dilution buffer:
Primary and secondary antibody should be diluted in 1x blocking solution to reduce non specific binding.
Coating antigen to microplate
Incubation with the antibody
Prepare a standard curve from the data produced from the serial dilutions with concentration on the x axis (log scale) vs absorbance on the Y axis (linear). Interpolate the concentration of the sample from this standard curve.