ELISA :
Coating of Plates :
Coat with 50 µl capture Antibody (usually it is monoclonal)
Over night 4 C
Dump over the sink
Block with 4% BSA (50 µl/well) and incubate 2 h in 37 C or better over night 4C
Main procedure :
Dump the blocking buffer out
Add 50 µl of Standard or diluted Sample / well in coated plates
Shake for1 h (RT)
Dump over the sink
Wash with 200 µl PBS
Add 50 µl Detection Antibody (usually polyclonal and biotin-labeled)
Incubate 1h (RT)
Wash 3x 10 min with 200 µl PBS/ 0.2% Tween
Wash 1x with 200 µl PBS
Add 50 µl HRP labeled SA , 30 min
(1 : 2500 in 4% BSA, ie. 2.4µl in 6 ml BSA)
Wash 3x 10 min PBS/Tween
Wash with 1x PBS (200µl)
Add 100µl/well Peroxidase substrate (TMB+H2O2 =1 :1) (Pierce Cat# :1854050 & 1854060)
Shake 2-10 min
Add 100 µl H2SO4 (0.18 M) to stop the reaction
Measure absorbance by 450 nm and 550 (background), subtract background
Software :
Go to X-Fluor
Load measurement parameter
HMGB1 ELISA.mps (because the last step (HRP) ist the same as other ELISAs we use)
For Using Titri
Save the Excel file as Text (Tab delimited) (*.txt)