Immunoprecipitation Troubleshooting

Immunoprecipitation

 

1. No antigen precipitated

 

  • Check that the antibody is listed as being suitable for IP.
  • Check the stringency of the washing step. Try using PBS instead of RIPA buffer as this may preserve the immune complex.
  • Check the compatibility of the primary antibody with Protein A or Protein G-PLUS agarose being used. For example, Protein A does not bind to goat IgG. If an inappropriate reagent is being used, switch to the correct product (see table below)

 

Binding Capacity, mg IgG/ml

 

Description

Specificity

Human

Mouse

Rat

Goat

Rabbit

Protein A Agarose

Mouse IgG2a, IgG2b & IgA
Rabbit polyclonals

12

8

4

0

12

Protein G PLUS-Agarose

Mouse IgG1 & IgG3
Rat IgG2a, IgG2b & IgG2c
Rabbit and goat polyclonals

12

8

8

8

10

Protein A/G PLUS-Agarose

All of the above antibodies

12

8

8

6

11

 

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