20
< BIOCHEMISTRY
International, Rochester, NY) for
1 hour at 37 °C. Wells were then rinsed
four times with 0.05% Tween 20 in
Ca
2+
- and Mg
2+
-free PBS (rinse/block
buffer), and 100 µL of primary anti-
body diluted 1:4000 in Superblock
(Pierce Chemical Co.) containing
0.05% Tween 20 (antibody dilution
buffer) was added to each well. After
1 hour of incubation at 37 °C, plates
were rinsed as above with rinse/block
buffer, 100 µL of secondary antibody
diluted 1:2000 in antibody dilution
buffer was added, and plates were incu-
bated for 1 hour at 37 °C. After incuba-
tion, plates were rinsed as above, 100 µL
of ABTS 1 STEP (Pierce Chemical Co.)
was added, and plates were incubated at
room temperature for 20 minutes. The
reaction was stopped by addition of
50 °C 2% SDS containing 0.5 mg/mL
Proteinase K, and plates were read
immediately in a SpectraMax 340 plate
reader (Molecular Devices, Corp.)
Summary :
After 1 hour of incubation at 37 °C, plates were rinsed as above with rinse/block buffer, 100 µL of secondary antibody diluted 1:2000 in antibody dilution buffer was added, and plates were incu- bated for 1 hour at 37 °C.
Tags :
plates,buffer,hour,added,rinsed,100,antibody,diluted,tween,dilution,005,rinseblock,containing