实验概要
This method involves coupling an IgG anti-IgM antibody to protein A or G beads (see method below). These beads can then be used in the normal immunopreciptiation procedure using the IgM antibody. The IgM will bind to the beads by binding to the anti-IgM antibody.
The procedure below describes how to couple the anti-IgM antibody to the protein A or G coated beads using dimethylpimelimidate (DMP).
主要试剂
1. Borate buffer 200 mM NaCl 3 M pH 9.0
2. Dimethylpimelimidate (DMP) 20 mM in 200 mM borate buffer 3 M NaCl, pH 9.0
3. Ethanolamine 200 mM pH 8 (1:80 dilution of stock ethanolamine)
4. Phosphate buffered saline (PBS)
5. PBS sodium azide 0.1%
6. Glycine 200 mM pH 2.5
7. Protein A or G beads (sepharose)
实验步骤
We recommend using 2 mg of antibody per ml wet beads (use appropriate antibody/protein A or G combination).
1. Add 250 mg Protein A or G sepharose beads (beads) to 2 ml PBS 0.1% sodium azide, for one hour before use. This allows the beads to swell.
2. Mix the beads with an appropriate concentration of antibody for 2 hours at room temperature (using a rotator or roller).
3. Centrifuge at 2500 rpm for 5 minutes.
4. Wash the beads twice in 10X volume of 200 mM borate buffer 3 M NaCl.
5. Resuspend the beads in 20 mM DMP.
6. Rotate / agitate the beads for 30 minutes at room temperature.
7. Centrifuge the beads at 2500 rpm for 5 minutes.
8. Wash the beads 2X in 200 mM borate buffer 3M NaCl.
9. Wash the beads 1X in 20 mM ethanolamine. This stops the coupling reaction.
10. Resuspend the beads in 20 mM ethanolamine and rotate for 2 hours at room temperature.
11. Centrifuge the beads at 2500 rpm for 5 minutes.
12. Wash the beads 2X in PBS.
13. Wash the beads 2X in 20 0mM glycine.
14. Wash the beads 2X in PBS.
15. The beads can be stored at 4oC in PBS 0.1% sodium azide (1 X PBS to 1 X beads)
16. These beads are now ready for use in immunoprecipitation with IgM antibody. From this point, proceed with the immunoprecipitation as described on our immunoprecipitation protocol page.