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Antibody standards for ChIP
Currently, there are a limited number of well-characterized antibodies against transcription factors and chromatin proteins. To ensure specificity of the antibodies used, the following tests must be employed. The data generated to characterize the antibody should be made publically available alongside the ENCODE dataset (see metadata standards).
Primary pathway to characterization All antibodies used for ChIP experiments should be characterized using immunoblotting OR immunofluorescence.
- Immunoblot Analysis. Successful antibodies should show essentially a single band of the predicted size by immunoblot analysis. The minimum standard to be met is that the predicted reactive band compose no less than one half of the total signal in the lane, as assessed by quantitative immunoblot analysis of immunoprecipitation using either nuclear extracts or whole cells (e.g. quantitative imaging of chemiluminescence or chemifluorescence).
- Immunofluorescence (IF) Analysis. If immunoblots are unsuccessful, immunofluorescence may be used as a characterization measure. The immunofluorescence pattern must conform to expectations (for example, nuclear staining for a chromatin protein or TF).
In addition, antibodies must be further characterized by the use of one of the four following methods:
- a. Knock-down of the target protein by genetic mutation , RNAi, or siRNA.
- i. siRNA or RNAi knockdowns should be conducted in duplicate.
- ii. For immunoblots, the band of the expected size along with additional immunoreactive bands should be reduced to 30% or less of the signal of unaffected cells.
- iii. For IF, any nuclear staining should be completely eliminated. Any cytoplasmic background remaining after genetic mutation, RNAi, or siRNA should be noted.
- b. Immunoprecipitation followed by mass-spec verification;
- i. Mass spectrometry of immunoprecipitated proteins extracted from major bands (or all material) separated though gel electrophoresis should identify peptides corresponding to the predicted target protein of the antibody. All immunoreactive bands identified by immunoblot analysis should be identified and shown to be that of the expected protein and/or not a known chromosomal associated protein. All proteins identified by MS should be reported.
- c. Immunoprecipitation with multiple antibodies against different parts of the target protein.
- i. It is expected that as the number of antibodies increase in the near future, the use of multiple antibodies against the same protein will become the standard for the field (see Appendix I). For antibodies generated against protein modifications, two independently raised antibodies are recommended, and antibodies should be thoroughly characterized to ensure that they recognize only the desired modification and not other related modifications using the minimum standards applied in Appendix II.
- ii. Each antibody may be used for ChIP experiments, and a statistically significant overlap of targets will be constitute characterization. Any reasonable method of correlation can be used (r2 greater or equal to 0.5) or 80% of the top 40% of the targets of one list should overlap that of the list from the second antibody (Appendix I). An alternative approach is to perform ChIP-chip or ChIP-seq for one antibody and to use a second antibody to test 24 targets selected from a range of positions using qPCR. At least 80% overlap of validated targets is expected. The full set of targets identified by each antibody, in addition to the intersection, should be reported.
- d. Immunoprecipitation with an epitope-tagged version of the protein.
- i. An epitope-tagged version of the target protein may be used, preferably driven by the endogenous gene promoter. Experiments should be conducted and analyzed as described above for the use of multiple antibodies.