Application
ELISA
Sample
Human Recombinant protein (Recombinant HBA1)
Specification
Recombinant HBA1
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Type
Indirect
Other product details
Dilution
1/200
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 1xPBS
Secondary antibody
Name
Dilution
1/5000
Additional data
Abcam response
Thank you for your valuable feedback and data from ELISA which has not been tested before with this antibody.
We would welcome any further Abreviews from customers who have tested this antibody on endogenous samples in ELISA.
We would welcome any further Abreviews from customers who have tested this antibody on endogenous samples in ELISA.
Additional Notes
Materials
- Recombinant HBA1, Abnova (Product# H00003039-Q01)
- 1xPBS coating buffer
- Rabbit polyclonal to Hemoglobin (ab92492)
- Donkey polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab98503)
- HRP substrate - SureBlue TMB Microwell Peroxidase Substrate, Insight Biotechnology Limited (Cat# 52-00-02)
- TMB Stop Solution, Insight Biotechnology Limited (Cat# 50-85-05)
Protocol
Coating antigen on microplate
1) Antigen (recombinant HBA1) was diluted to 2µg/ml, 0.2µg/ml and 0.02µg/ml in 1xPBS. Wells of a microtiter were coated with 50µl of antigen.
2) The plate was sealed with an adhesive plastic and incubate @ 4°C overnight.
3) Following incubation, coating solution was removed and the plate washed (x3) with 200µl PBS/0.05% Tween-20 (PBS-T).
Blocking
1) The remaining protein-binding sites were blocked by adding 200µl blocking buffer (5% BSA in 1xPBS).
2) The plate was sealed and incubated for 2 h @ 22°C.
3) Following incubation, blocking solution was removed and the plate washed (x2) with 200µl PBS-T.
Incubation with primary antibody
1) 100µl of diluted primary antibody (1/200, 1/400 and 1/800 in 1xPBS) was added to different wells of the plate, the plate was sealed and incubated for 1 hour @ room temperature.
2) Following incubation, antibody solution was removed and the plate washed (x4) with 200µl PBS-T.
Incubation with secondary antibody
1) 100µl of diluted secondary antibody (1/5000 in 5%BSA/1xPBS) was added to the plate, the plate was sealed and incubated for 1 hour @ 22°C.
2) Following incubation, antibody solution was removed and the plate washed (x4) with 200µl PBS-T.
Detection
1) Detection was performed using the HRP substrate - SureBlue TMB Microwell Peroxidase Substrate.
2) 100µl of substrate was added to each well, as colour developed (blue colour) the plate was gently tapped.
3) After 20-25 minutes the reaction was stopped by addition of 100µl of TMB Stop Solution.
4) The plate was read on a microplate reader using a 450nm filter.
- Recombinant HBA1, Abnova (Product# H00003039-Q01)
- 1xPBS coating buffer
- Rabbit polyclonal to Hemoglobin (ab92492)
- Donkey polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab98503)
- HRP substrate - SureBlue TMB Microwell Peroxidase Substrate, Insight Biotechnology Limited (Cat# 52-00-02)
- TMB Stop Solution, Insight Biotechnology Limited (Cat# 50-85-05)
Protocol
Coating antigen on microplate
1) Antigen (recombinant HBA1) was diluted to 2µg/ml, 0.2µg/ml and 0.02µg/ml in 1xPBS. Wells of a microtiter were coated with 50µl of antigen.
2) The plate was sealed with an adhesive plastic and incubate @ 4°C overnight.
3) Following incubation, coating solution was removed and the plate washed (x3) with 200µl PBS/0.05% Tween-20 (PBS-T).
Blocking
1) The remaining protein-binding sites were blocked by adding 200µl blocking buffer (5% BSA in 1xPBS).
2) The plate was sealed and incubated for 2 h @ 22°C.
3) Following incubation, blocking solution was removed and the plate washed (x2) with 200µl PBS-T.
Incubation with primary antibody
1) 100µl of diluted primary antibody (1/200, 1/400 and 1/800 in 1xPBS) was added to different wells of the plate, the plate was sealed and incubated for 1 hour @ room temperature.
2) Following incubation, antibody solution was removed and the plate washed (x4) with 200µl PBS-T.
Incubation with secondary antibody
1) 100µl of diluted secondary antibody (1/5000 in 5%BSA/1xPBS) was added to the plate, the plate was sealed and incubated for 1 hour @ 22°C.
2) Following incubation, antibody solution was removed and the plate washed (x4) with 200µl PBS-T.
Detection
1) Detection was performed using the HRP substrate - SureBlue TMB Microwell Peroxidase Substrate.
2) 100µl of substrate was added to each well, as colour developed (blue colour) the plate was gently tapped.
3) After 20-25 minutes the reaction was stopped by addition of 100µl of TMB Stop Solution.
4) The plate was read on a microplate reader using a 450nm filter.
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投稿 Mar 07 2011