Biology Reference
In-Depth Information
15.5.1.3
Procedure
Buffer exchange and concentration of antibody. Dilute 2 mL (20 mg) 1D4 mAb
with 12 mL PBS. Concentrate solution with centrifugal ultrafiltration device
(100 kDa Amicon Ultra-15, at RT with 2000
g
) down to 0.4 mL volume.
Repeat dilution and concentration two more times. Test the concentration of the
concentrated sample by UV-Vis spectroscopy. Typically, more than 85% protein
is recovered.
Periodate oxidation of antibody. Dilute antibody concentrate to 20 mg/mL
with PBS. Add 1 part 200 mM NaIO
4
(final concentration 10 mM) to 35 parts of
the antibody solution, mix well, and incubate protected from light for 15 min
at RT.
Stop reaction with 50
m
L 400 mM Na
2
SO
3
(final concentration 20 mM).
Add 100
L 50 mM biocytin hydrazide (final concentration 5 mM) and mix on a
rotating wheel for 2 h at RT.
Cool sample on ice and add 100
m
L 300 mM cyanoborohydride (final
concentration 30 mM). Incubate on ice for 40 min.
Dialyze sample in 10 kDa Slide-A-Lyzer against prechilled 500 mL PBS
supplemented with 55 mL glycerol
m
1.11 mL 10% NaN
3
(final concentration
0.02%) at 4
C. Exchange dialysis buffer twice every 4-8 h.
Determine the concentration of the dialyzed sample by UV-Vis spectroscopy.
Typically, we find better than 50% overall yield. The sample can be stored at 4
C
for up to 3 months without significant loss of activity.
þ
15.5.2
Method 2: biotinylation of antibodies with
sulfo-NHS-SS-biotin
15.5.2.1
Materials
Disposable column, 10 mL (Pierce 29924)
Sephadex G50, fine (GE) 50% slurry in 20% ethanol
2 mL 10 mg/mL 1D4 mAb (in PBS with 10% glycerol 0.02% NaN
3
, stored
at
80
C)
Sulfo-NHS-SS-biotin (Pierce 21328; MW
¼
606.69 g/mol)
15.5.2.2
Solutions and buffers
10 mM SULFO-NHS-SS-BIOTIN:
1 mg sulfo-NHS-SS-biotin.
164
L purified water.
Prepare immediately before use.
m
78% GLYCEROL:
10 mL purified water.
35 mL glycerol.
Adjust volume to 45 mL with water.
Filter with a 0.2
20
C.
m
m Steriflip unit and store at
