Immunoprecipitation
From Diseasepedia
No4. Immunoprecipitation
Summary
Determines antigen presence, quantity, size, turnover, modifications, associations
Multiple steps over 1/2 to 1 day
Semi-quantitative to quantitative
Sensitivity dependent on relative quantity of antigen and affinity of antibody
Needs high affinity/ avidity antibody
1. Add 50 ul of normal rabbit serum per 1.0 ml of antigen solution.
2. Incubate 1 hour on ice.
3. During this incubation, wash fixed S.aureus Cowan (SAC) once in lysis buffer. Use 100 ul of packed SAC per 50ul of normal rabbit serum. It is easiest to spin SAC briefly at 10,000g for 30 seconds and resuspend the pelleted SAC by triturating with a pasteur pipet. Spin again at 10,000g and remove the wash buffer. Set the packed pellet of washed SAC aside on ice.
4. After the 1-hour incubation, use the lysaste to resuspend the washed pellet of SAC.
5. Incubate 30 minutes on ice.
6. Spin at 10,000g for 15 minutes at 4°C. Carefully remove and save the supernatant.
7. Divide the supernatant and aliquot into the appropriate number of 1.5 ml conical tubes. To invidual tubes, add the immune or control serum (1ul), hybridoma tissue-culture supernatant (50ul), or ascites fluid (1ul). Over volumes will be appropriate in specific settings as determined by titration of antibody volume versus a constant antigen amount.
8. Incubate on ice for 1 hour.
9. To the antibody-antigen reaction, add protein A or protein G beads depending on the type of antibody used. Add 100 ml of a 10% vol/vol slurry made in lysis buffer (final bead volume will be 10 ml). Close the caps securely. Incubate 1 hour at 4°C will rocking.
10. Collect the beads by centrifugation at 10,000g for 15 seconds at 4°C. Wash the immune complexes three times with lysis buffer. The lysate and wash buffers are easily removed by aspiration through a 23-gauge needle.
11. Remove the final wash as completely as possible.
12. Use the immune complexes for the appropriate assay, often SDS-polyacrylamide elextrophoresis.
| Antibody source | Protein A |
Protein G |
| Monoclonal antibodies mouse IgG1 Mouse IgG2a,IgG2b,IgG3 rat |
V |
V |
|
Polyclonal antibody |
V V V V V V V |
V |
