Team:USTC CHINA/Notebook/Protocols/ELISA
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<h1>ELISA</h1> | <h1>ELISA</h1> | ||
<p>ELISA | <p>ELISA | ||
- | A. Antigen Coating | + | A. Antigen Coating </br> |
- | 1. Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer (coating buffer), e.g. human IgG (0.025mg/ml) in coating buffer. | + | 1. Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer (coating buffer), e.g. human IgG (0.025mg/ml) in coating buffer.</br> |
- | 2. Pipette 0.2 ml of the above solution to each well of the microtiter plate. | + | 2. Pipette 0.2 ml of the above solution to each well of the microtiter plate.</br> |
- | + | 3. Incubate at 37 0C for 30 min. </br> | |
- | 4. Remove the coating solution. Wash three times with PBS-T (0.2 ml/well). | + | 4. Remove the coating solution. Wash three times with PBS-T (0.2 ml/well). </br> |
- | 5. Blocking step: 0.5% BSA-PBS (0.2ml/well) at 370C for 30 min (an additional blocking step may be required to block non-specific binding). | + | 5. Blocking step: 0.5% BSA-PBS (0.2ml/well) at 370C for 30 min (an additional blocking step may be required to block non-specific binding). </br> |
- | B. Primary Antibody Reaction | + | B. Primary Antibody Reaction </br> |
- | 1. Dilute the primary (first) antibodies in PBS-T, e.g. Rabbit-anti-human IgG antiserum (different dilutions of antiserum, 1:400~1:51200). | + | 1. Dilute the primary (first) antibodies in PBS-T, e.g. Rabbit-anti-human IgG antiserum (different dilutions of antiserum, 1:400~1:51200). </br> |
- | 2. Add 0.2 ml of the diluted first antibody to each well. Negative control should be included (No first Ab.). | + | 2. Add 0.2 ml of the diluted first antibody to each well. Negative control should be included (No first Ab.). </br> |
- | 3. Incubate at 37 0C for 1 hour. | + | 3. Incubate at 37 0C for 1 hour. </br> |
- | 4. Wash three times with PBS-T (0.2 ml/well). | + | 4. Wash three times with PBS-T (0.2 ml/well). </br> |
- | C. Application of Secondary Antibody | + | C. Application of Secondary Antibody </br> |
- | 1. Dilute the peroxidase conjugated secondary antibody in PBS-T, e.g. | + | 1. Dilute the peroxidase conjugated secondary antibody in PBS-T, e.g. Goat-anti-rabbit IgG-HRP (1:20,000). Add 0.2 ml of this solution to each well. </br> |
- | Goat-anti-rabbit IgG-HRP (1:20,000). Add 0.2 ml of this solution to each well. | + | 2. Incubate at 37 0C for 30 min. </br> |
- | 2. Incubate at 37 0C for 30 min. | + | 3. Wash three times with PBS-T (0.2 ml/well). </br> |
- | 3. Wash three times with PBS-T (0.2 ml/well). | + | D. Substrate Preparation </br> |
- | D. Substrate Preparation | + | 1. During the last incubation and immediately before use, dissolve o-Phenylenediamine in Citric acid-sodium hydrogen phosphate buffer, add 30% H2O2 before use. </br> |
- | 1. During the last incubation and immediately before use, dissolve | + | E. Development </br> |
- | o-Phenylenediamine in Citric acid-sodium hydrogen phosphate buffer, add 30% H2O2 before use. | + | 1. Add 0.2 ml of the freshly prepared substrate to each well. </br> |
- | E. Development | + | 2. Orange-yellow color should develop in positive wells after 5 minutes.</br> |
- | 1. Add 0.2 ml of the freshly prepared substrate to each well. | + | 3. Stop reaction with 50 l per well of preferred stopping reagent and read at 490nm in a microplate reader.</br> |
- | 2. Orange-yellow color should develop in positive wells after 5 minutes. | + | REAGENTS </br> |
- | 3. Stop reaction with 50 l per well of preferred stopping reagent and read at 490nm in a microplate reader. | + | 1. Coating buffer (Carbonate-Bicarbonate buffer, pH 9.6) : Na2CO3 0.15g, NaHCO3 0.293g, add H2O to 100ml (pH 9.6).</br> |
- | REAGENTS | + | 2. Phosphate buffered saline (PBS): NaCl 8g, KCl 0.2g, KH2PO4 0.24g,Na2HPO4. 12 H2O 2.9g, add H2O to 1000ml (pH 7.4).</br> |
- | 1. Coating buffer (Carbonate-Bicarbonate buffer, pH 9.6) : Na2CO3 0.15g, NaHCO3 0.293g, add H2O to 100ml (pH 9.6). | + | 3. Washing buffer (PBS-T): PBS + 0.05% Tween 20.</br> |
- | 2. Phosphate buffered saline (PBS): NaCl 8g, KCl 0.2g, KH2PO4 0.24g,Na2HPO4. 12 H2O 2.9g, add H2O to 1000ml (pH 7.4). | + | 4. First antibody: Rabbit-ani-human IgG antiserum.</br> |
- | 3. Washing buffer (PBS-T): PBS + 0.05% Tween 20. | + | 5. Peroxidase conjugated secondary antibody: Goat-anti-rabbit IgG-HRP (1:20,000).</br> |
- | 4. First antibody: Rabbit-ani-human IgG antiserum. | + | 6. Substrate: substrate buffer(fresh):</br> |
- | 5. Peroxidase conjugated secondary antibody: Goat-anti-rabbit IgG-HRP (1:20,000). | + | 0.1M Citric acid (2.1g/100ml), 6.1ml;</br> |
- | 6. Substrate: substrate buffer(fresh): | + | 0.2M Na2HPO4. 12 H2O(7.163g/100ml), 6.4ml;</br> |
- | 0.1M Citric acid (2.1g/100ml), 6.1ml; | + | ddH2O 12.5ml;</br> |
- | 0.2M Na2HPO4. 12 H2O(7.163g/100ml), 6.4ml; | + | dissolve 8mg o-Phenylenediamine;</br> |
- | dissolve 8mg o-Phenylenediamine | + | add 40ml of 30% H2O2 before use.</br> |
- | 7. Stopping reagent: 2M H2SO4 | + | 7. Stopping reagent: 2M H2SO4</br> |
</p></div> | </p></div> |
Revision as of 14:48, 25 September 2013