This immunoprecipitation protocol should be used as a guide for each researcher to build their own protocol, as each reagent will need to be optimized for use in the particular species, tissue type and application combination.
Tris Buffered Saline. Use a 10x TBS, pH 7.5 (1.0M Tris HCl, 1.5M NaCl). Dilute appropriate volume to 1x with de-ionized water. Store at room temperature up to one month.
Lysis Buffer. TBS containing 1.0% of an appropriate detergent, 1mg.ml bovine serum albumin BSA, and an appropriate proteinase inhibitor.
Dilution Buffer. Same as lysis buffer without proteinase inhibitor.
Agarose conjugates for lysate pre-treatment. Pre-absorb lysates to remove non-specific binding to primary and secondary antibodies. Use agarose normal IgG from the same species as the primary antibody and the host secondary antibody. Prepare washed slurry at 1:1 using dilution buffer.
Control for primary antibody. For polyclonal antiserum, use non-immune serum from the same species. For monoclonal antibodies, use the same isotype and purity.
Agarose conjugates for Immunoprecipitation. Use agarose secondary antibody conjugate against the same species as the primary antibody. Prepare washed slurry at 1:1 using dilution buffer.
Tris Buffer. Prepare 0.05M Tris buffer, pH 6.8.
2x SDS-PAGE Sample Buffer
Prepare lysate by incubating 5 x 107 cells in lysis buffer for 30-60 minutes on ice.
Vortex lysate and centrifuge for 10 minutes at 250 x g to remove nuclei. Retain supernate.
Clarify supernatant by centrifugation for 30 minutes at 100,000 x g or microcentrifuge for 30 minutes at 10,000 x g.
Pre-treat lysate to remove nonspecific protein binding by adding agarose conjugate. Use 10µl of control agarose per 200 µl lysate. Shake for 1 hour at 4ºC. Centrifuge at 200 x g. Save supernatant.
Add 200 µl of pretreated lysate containing antigen to each of two microfuge tubes. Bring volume to 1 ml with dilution buffer.
Add primary antibody to one tube. For polyclonal antiserum or ascites fluid use 0.5-5 µl. For tissue culture supernatant, use 10-100 µl. To the second tube, add an equivalent volume of control for primary antibody. Incubate on ice for one hour.
For immunoprecipitation add 50µl of agarose conjugate per tube. Mix with gentle shaking for 1 hour at 4ºC.
Centrifuge tube 1 minute at 200 x g or micro-centrifuge for 5 seconds. Carefully remove the supernatant with a pipette. Gently resuspend pellet in 1 ml dilution buffer. Repeat wash. Follow with a wash in TBS and then a final wash in 0.5 M Tris, pH 6.8.
Centrifuge again as above. Add 20-50 µl of sample buffer. Mix and heat for 5 minutes at 100 ºC. Micro-centrifuge briefly and apply supernatant directly to non-reducing SDS-PAGE. If reducing conditions are desired, transfer the supernate to a new tube and add 5% 2-mercaptoethanol. Mix and heat as above.
Electrophorese protein mixture. Stain gel or immunoblot (using our Western Blot Protocol) to visualize. Bands present will include polypeptides of antigen and antibodies used.
How to choose the Correct Beads:
|Species immunoglobulin isotype||Protein A||Protein G|
|Human IgG1||Strong Binding||Strong Binding|
|Human IgG2||Strong Binding||Strong Binding|
|Human IgG3||No Binding||Strong Binding|
|Human IgG4||Strong Binding||Strong Binding|
|Human IgM||Use anti human IgM|
|Human IgE||No Binding||Weak Binding|
|Human IgA||No Binding||Weak Binding|
|Mouse IgG1||Weak Binding||Strong Binding|
|Mouse IgG2a||Strong Binding||Strong Binding|
|Mouse IgG2b||Medium Binding||Medium Binding|
|Mouse IgG3||Weak Binding||Weak Binding|
|Mouse IgM||Use anti Mouse IgM|
|Rat IgG||No Binding||Weak Binding|
|Rat IgG2a||No Binding||Strong Binding|
|Rat IgG2b||No Binding||Medium Binding|
|Rat IgG2c||Weak Binding||Medium Binding|
|Chicken all isotypes||No Binding||No Binding|
|Cow all isoptypes||Medium Binding||Strong Binding|
|Goat all isotypes||No Binding||Medium Binding|
|Guinea Pig all isotypes||Strong Binding||Medium Binding|
|Hamster all isotypes||Weak Binding||Medium Binding|
|Horse all isotypes||Weak Binding||Strong Binding|
|Pig all isotypes||Weak Binding||Medium Binding|
|Rabbit all isotypes||Strong Binding||Medium Binding|
|Sheep all isotypes||No binding||Strong Binding|