Labelling Pax6 with Dig-NHS

Procedure

Pax6 AB is at 5 mg/ml, 1 ml

1) Dialyze 500 µl into PBS O/N then one change in morning 2 hours.

2) Spin antibody and check protein concentration by BioRad

STD OD 595

0

1

3

6

9

1 µl Pax6

3) Bring antibody to approx. 2.5 mg/ml in PBS. Final vol:

4) Check 1M NaHCO3 solution for pH (should be pH 8.3)

5) Add 1/10 volume of 1M NaHCO3.

6) Remove some Pax6 AB as non-labelled control.

7) Thaw NHS-Dig and spin briefly. Add Dig-NHS (10 mg/ml stock in DMSO) at 1:50 dilution to AB

(20 µl per 1 ml) and mix rapidly

(Dig is stored in liq. Nitrogen, tower 6 box H)

8) Incubate 1 hour at RT with occasional mixing

9) Add 100 mM Glycine pH 7 (30 µl per 500 µl of AB) incubate atleast 10 min.

10) Dialyze labeled and unlabeled sample into TBS O/N

11) Store with 0.1% BSA in 50% glycerol at -20 C.