Labelling Pax6 with Dig-NHS: Difference between revisions
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== Procedure == | |||
Pax6 AB is at 5 mg/ml, 1 ml | Pax6 AB is at 5 mg/ml, 1 ml | ||
Latest revision as of 10:33, 27 March 2015
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.