ELISA Protocol for a 96-well Plate: Difference between revisions
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Elisa protocol for one 96-well plate | Elisa protocol for one 96-well plate | ||
Latest revision as of 16:18, 15 January 2013
Elisa protocol for one 96-well plate to prepare:
70ml blotto
50 ml-PBS
10 mM phosphate, pH7 0.5 ml of 1 M stock 5.5 ml 1 step ultra TMB developing solution 5 ml-2 M H2SO4 450 ul- 5ug/ul K57/1
5ml-1/10000 KPL horseradish peroxidase in blotto
Notes: Protocol: 1)Coat plates 2) Remove antigen liquid. 3) Block nonspecific protein binding sites. 4) 1° antibody incubation. 5) Remove antibody dilutions. To wash, add 150 µl of Blotto per well. Rock for 5 min at room temp. Remove wash solution by dumping. Repeat washes two times. 6) After last wash, add 50 µl of KPL horseradish peroxidase (KPL HRP GxM H&L) conjugated second antibody, diluted in blotto. Rock for 45 min at room temp. 7) Remove HRP-conjugated solution. To wash add ≥150 µl of PBS (0.15 M NaCl, 10 mM phosphate, pH7) per well. Rock for 5 min at room temp. Remove wash solution by dumping. Repeat washes two times. 8) To develop, add 50 µl of developing solution per well. Allow plates to develop on rocker at room temp ~5 minutes until sufficient blue color develops then stop developing with 50 µl 2 M H2SO4 9) Read Abs450 on Hell plate reader, record final values in excel spreadsheet |