B-Gal ELISA

After trypsin and resuspending in media
Spin and resuspend in 50ul PBS
Used 35ul from the 50ul and add 100ul lysis buffer (dilute from 5x B-gal lysis buffer, Soln 8)
Rotate at RT 20min
Store at -80C
Spin max speed 15min 4C to remove cell debris
Use supernatant for ELISA

Make standard
12.5ul of 38ng/ml B-gal enzyme stock solution (Soln 1 -20C) in 500ul of sample buffer (Soln 7, 4C) = 950pg/ml
Serial dilution down to 15pg/ml (250ul + 250ul)
Keep blank

Pipette 200ul of standard dilutions or 200ul of samples into wells (I use as much embryo samples as I can to get good signal), make up to 200ul with sample buffer
Seal with adhesive film, incubate 1h at 37C
Dilute washing buffer from 10x (Soln 6, 4C)

Wash 3x 30s with 1 washing buffer 250ul each
Dilute Anti-B-Gal-DIG solution (Soln 2, -20C) 100x from 50ug/ml to 0,5ug/ml in sample buffer
Pipette 200ul of diluted anti-Bgal-DIG into wells
Seal, incubate 1h 37C

Wash 3x30s
Dilute Anti-DIG-POD (Soln 3, 4C) 133x from 20U/ml to 150mU/ml in sample buffer
Pipette 200ul diluted anti-DIG-POD into wells
Seal, incubate 1h 37C

Wash 3x30s
Add 1mg of substrate enhancer (Bottle 5, 4C) per ml of ABTS substrate solution (Soln4, 4C)
Mix by stirring for 30min RT
Pipette 200ul of POD substrate (Soln 4+5) into wells
Incubate RT 15-40min, shake

Measure absorbance at 405nm with a plate reader. Colour stable for a few hours.