Primary antibody:
Thermo-Fisher MA523515 (Rabbit)
Source material:
Cell line: HEK293
Secondary antibody:
EMD Millipore 12-349 (Goat)
Lysis Conditions:
RIPA buffer
at
22°C
for
1 min
Gel Conditions
Run conditions:
200 V / 0 Amps for 0.5 hr
Reducing conditions:
Non-reducing
Transfer Conditions
Membrane type:
PVDF
Transfer method:
Wet
Transfer buffer:
Tris-glycine with 10% methanol
Transfer voltage:
100 V
Transfer current:
0 Amps
Transfer temperature:
4°C
Transfer duration:
1 hr
Blocking
Blocking buffer:
2% milk, 0% BSA, No serum
Blocking temperature:
22°C
Duration:
1 hr
Primary Incubation
Antibody 1 dilution
1:1,000
Incubation temperature
22°C
Incubation duration
1 hr
Number of washes
3
Wash buffer
TBST, 0.1% Tween-20
Wash duration
5 min
Secondary Incubation
Antibody 2 dilution
1:20,000
Incubation temperature
22°C
Incubation duration
1 hr
Number of washes
3
Wash buffer
TBST, 0.1% Tween-20
Wash duration
5 min
Detection
Detection method
Enhanced chemiluminescence (ECL)
Expected band size
97kDa
Result:
No specific bands
Background:
High background
Related Publications
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Record 3526 author: Amber Morey
Last edit: Thu Feb 23, 2023
Stripped blot, lots of residual antibody from first attempt to probe