Antibody Characterization StandardsENCODE 3February 2014Characterization of an antibody for a transcriptional regulator (including TFs, chromatinremodelers, and chromatin modifiers)Before ChIP-seq data can be submitted for any transcriptional regulator (including TFs, RNAbinding proteins and chromatin remodelers), the specific lot number of the antibody used tocollect the data must be approved for use in that cell type by the antibody characterizationcommittee of the ENCODE DCC. A primary and secondary characterization must both besubmitted for review and deemed COMPLIANT, in order for an antibody lot to be approved foruse.
1. In all cases, a primary characterization of the antibody must be provided for each lotnumber and each specific cell type. This can be either an immunoblot (Western blot) or animmunoprecipitation (IP-Western). [slide 1]a. If the major band is within 20% of the size predicted by the size of the coding region ofthe protein and corresponds to >50% of all bands on the gel (excluding the antibody bands inthe case of an immunoprecipitation), then the antibody passes this initial characterization. Eachimmunoblot should indicate which cells types were tested and include size markers. Forimmunoprecipitations, a control IgG precipitation should also be included.
b. If the antibody does not pass this initial characterization (e.g. due to multiple splicevariants or multiple modifications that alter the electrophoretic properties of the protein), it canbe rescued by a secondary characterization that supports the conclusion that the band(s)detected correspond to the correct protein (e.g. all bands are reduced upon treatment withsiRNA to that protein).
2. Use of the same lot number of a previously characterized antibody in a new cell type.
If a specific lot number for an antibody has previously passed both primary and secondarycharacterization in another cell type and if the banding pattern on the immunoblot orimmunoprecipitation is the same in the new cell type as in the characterized cell type, then nofurther characterization is needed for the antibody in that new cell type. If the banding pattern isdifferent in the new cell type, a secondary characterization must be performed in the new celltype. [slide 2]3. Use of a new lot number of a previously characterized antibody. If this is the first timethat a new lot number has been used for a previously characterized antibody, a primarycharacterization must be performed. However, if the pattern is the same as for the old lotnumber of the previously characterized antibody, then no further characterization is required. Ifthe banding pattern is different, a secondary characterization must be performed. [Slide 3]Allowable Secondary Characterization Methods:1) Secondary characterization using siRNA or shRNA against the mRNA of the targetprotein. For siRNA or shRNA characterization, the band(s) detected by the antibody should bereduced to no more than 50% of the original signal. The sequence or vendor and catalogAntibody	  Characterization,	  ENCODE	  3	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  February	  2014	  1	  	  number of the oligonucleotide(s) should be provided. A control knockdown should also beperformed. Cell types should be labeled and size markers should be included on theimmunoblot. A brief description of the transfection protocol should also be provided.
2) Secondary characterization using ChIP-seq data obtained using a previouslycharacterized antibody for that factor. If ChIP-seq data for a different lot number of apreviously characterized antibody or a previously characterized, but different, antibody for agiven transcriptional regulator is available, this ChIP-seq data can be used for characterizationof a new antibody or new lot number. In addition, ChIP-seq data obtained using an epitopetagged version of the target protein and an antibody that recognizes the tag can be used forcomparison. The ChIP-seq data from the new antibody or new lot number should be comparedto the previous ChIP-seq data. If the two datasets pass the ENCODE IDR cuts-offs for narrowpeak ChIP-seq reproducibility (see below for current IDR standards), then the secondarycharacterization of the new antibody/lot number is compliant with this defined standard.
3) Secondary characterization using overexpressed, tagged proteins. For proteins thatare resistant to knockdown using siRNAs (e.g. very stable proteins), comparison tooverexpressed, tagged proteins can be used for characterization. In this case, the primarycharacterization of the antibody must first show the appropriate specificity. Then, two side-byside immunoblots should be performed using control cells and cells overexpressing the factor.
The first immunoblot should employ the antibody to the tag to show the position of theexogenous factor and the second immunoblot should employ the antibody to the endogenousfactor to show that the band in the control cells that is identified by the endogenous antibody isthe same size as the exogeneous protein.
Notes:• These methods refer to characterization of antibodies that recognize endogenouslyexpressed proteins. The requirements for characterization of epitope-tagged proteinsare described elsewhere.
• It was generally agreed that the method of immunoprecipitation followed by massspectrometry would be appropriate for use as a secondary characterization. However,the details of the requirements for this method have not yet been finalized.
• At the current time, motif enrichment in the ChIP-seq peaks is not considered a validantibody characterization method.
• Current IDR standards for a narrow-peak ChIP-seq dataset are:Rescue Ratio RR_new = | Np U Nt | / | Np ^ Nt |Self consistency ratio SR_new = | N1 U N2 | / | N1 ^ N2 |where^ = intersection (common) of 2 peak setsU = union (merge) of 2 peaks setsIf (RR_new > 2) AND (SR_new > 2) then the replicates are proclaimed to have lowreproducibility (failed) and flagged with -1 quality scoreIf (RR_new > 2) OR (SR_new > 2) but not both, then the replicates are proclaimed tohave moderate reproducibility (passed) and flagged with a 0 quality scoreIf (RR_new <= 2) AND (SR_new <= 2) then the replicates are proclaimed to have highreproducibility (passed) and flagged with +1 quality scoreAntibody	  Characterization,	  ENCODE	  3	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  February	  2014	  2	  	  Characterization of an antibody for an RNA Binding ProteinBefore binding data can be submitted for any RNA binding protein (RBP), the specific lotnumber of the antibody used to collect the data must be approved for use in that cell type bythe antibody characterization committee of the ENCODE DCC. A primary and secondarycharacterization must both be submitted for review and deemed COMPLIANT, in order for anantibody lot to be approved for use.
1. In all cases, a primary characterization of the antibody must be provided for each lotnumber and each specific cell type. This can be either an immunoblot (Western blot) or animmunoprecipitation (IP-Western). [slide 1]a. If the major band is within 20% of the size predicted by the size of the coding region ofthe protein and corresponds to >50% of all bands on the gel (excluding the antibody bands inthe case of an immunoprecipitation), then the antibody passes this initial characterization. Eachimmunoblot should indicate which cells types were tested and include size markers. Forimmunoprecipitations, a control IgG precipitation should also be included.
b. If the antibody does not pass this initial characterization (e.g. due to multiple splicevariants or multiple modifications that alter the electrophoretic properties of the protein), it canbe rescued by a secondary characterization that supports the conclusion that the band(s)detected correspond to the correct protein (e.g. all bands are reduced upon treatment withsiRNA to that protein).
2. Use of the same lot number of a previously characterized antibody in a new cell type.
If a specific lot number for an antibody has previously passed both primary and secondarycharacterization in another cell type and if the banding pattern on the immunoblot orimmunoprecipitation is the same in the new cell type as in the characterized cell type, then nofurther characterization is needed for the antibody in that new cell type. If the banding pattern isdifferent in the new cell type, a secondary characterization must be performed in the new celltype. [slide 2]3. Use of a new lot number of a previously characterized antibody. If this is the first timethat a new lot number has been used for a previously characterized antibody, a primarycharacterization must be performed. However, if the pattern is the same as for the old lotnumber of the previously characterized antibody, then no further characterization is required. Ifthe banding pattern is different, a secondary characterization must be performed. [Slide 3]Allowable Secondary Characterization Methods:1) Secondary characterization using siRNA or shRNA against the mRNA of the targetprotein. For siRNA or shRNA characterization, the band(s) detected by the antibody should bereduced to no more than 50% of the original signal. The sequence or vendor and catalognumber of the oligonucleotide(s) should be provided. A control knockdown should also beperformed. Cell types should be labeled and size markers should be included on theimmunoblot. A brief description of the transfection protocol should also be provided.
2) Secondary characterization using overexpressed, tagged proteins. For proteins thatare resistant to knockdown using siRNAs (e.g. very stable proteins), comparison toAntibody	  Characterization,	  ENCODE	  3	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  February	  2014	  3	  	  overexpressed, tagged proteins can be used for characterization. In this case, the primarycharacterization of the antibody must first show the appropriate specificity. Then, two side-byside immunoblots should be performed using control cells and cells overexpressing the factor.
The first immunoblot should employ the antibody to the tag to show the position of theexogenous factor and the second immunoblot should employ the antibody to the endogenousfactor to show that the band in the control cells that is identified by the endogenous antibody isthe same size as the exogeneous protein.
Notes:• These methods refer to characterization of antibodies that recognize endogenouslyexpressed proteins. The requirements for characterization of epitope-tagged proteinsare described elsewhere.
• It was generally agreed that the method of immunoprecipitation followed by massspectrometry would be appropriate for use as a secondary characterization. However,the details of the requirements for this method have not yet been finalized.

Antibody	  Characterization,	  ENCODE	  3	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  February	  2014	  4	  	  Characterization of a Histone AntibodyBefore ChIP-seq data can be submitted for any antibody that detects a modified histone, thespecific lot number of the antibody used to collect the data must be approved by the antibodycharacterization committee of the ENCODE DCC. A primary and secondary characterizationmust both be submitted for review and deemed COMPLIANT, in order for an antibody lot to beapproved for use. [slide 4]1. In all cases, a primary characterization of each lot number of an antibody for amodified histone must be performed. Primary characterization of a specific lot number for ahistone antibody should include a immunoblot using nuclear or whole cell extract (to show apositive band at the size of the histone) plus a recombinant unmodified version of that histone(to show lack of reactivity with the unmodified histone). Extracts from at least 3 cell lines/typesshould be analyzed, including the species in which the antibody will be used. The specifichistone band detected in the extracts should constitute at least 50% of the protein signal andshow at least 10-fold enrichment relative to any other single band. In addition, this signal shouldbe at least 10-fold enriched relative to that detected using the unmodified recombinant histone.
Each immunoblot should label the cells and include size markers.
2. Secondary characterization using dot blots, peptide arrays or peptide competitions.
Commercial arrays are acceptable and the vendor and catalog number should be provided. Ifarrays that include small numbers of peptides are used, it is critical that the array contain themost relevant peptides for the antibody being tested. If peptide competition is used to validate ahistone antibody, it is also important that the most relevant peptides for the antibody beingtested are used in the competition series. A 10-fold enriched binding signal for the modificationof interest relative to other modifications is required. An annotated map of the peptides presenton the blot or array should be provided.
3. Additional cell types. Once a specific lot number for an antibody to a modified histone haspassed both the primary and secondary characterizations, it is eligible for use in all cell types(with the caveat that the species must have been included in the primary characterization).
Note. It was generally agreed that it should be allowable to use similarity of ChIP-seq data of adifferent lot number of the same histone antibody, a different antibody for the same histonemodification, or a component of the histone modifying complex that puts on the histone mark asa characterization method. However, a quantitative measure of how closely the patterns mustmatch has not yet been decided; these quantitative measures may not be the same for allhistone marks. Once quantitative measures have been determined, this characterizationmethod may be allowed.

Antibody	  Characterization,	  ENCODE	  3	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  February	  2014	  5	  Ini$al	  characteriza$on	  of	  an	  an$body	  (for	  TFs,	  chroma$n	  remodelers,	  chroma$n	  modiﬁers,	  RBPs)	  	   for	  all	  lot	  numbers	  of	  any	  an5body	  A	  Western	  blot	  or	  IP-­‐Western	  must	  be	  done	  using	  extracts	  from	  the	  same	  cell	  type	  used	  for	  the	  ChIP-­‐seq	  	  Major	  band	  is	  within	  20%	  of	  the	  right	  size	  and	  >50%	  signal	  on	  gel	  Mul5ple	  splice	  variants,	  heavy	  modiﬁca5ons	  Meets	  requirements	  Not	  Pass	  Secondary	  characteriza5on:	  Conﬁrm	  with	  siRNA	  	  siRNA,	  tagging,	  ChIP-­‐seq#,	  Mass	  Spec*	  Meets	  requirements	  #not	  appropriate	  for	  RBP	  *not	  yet	  approved	  Antibody Characterization, ENCODE 3wrong	  size,	  ladder	  of	  bands,	  no	  signal	  Does	  not	  meet	  requirements	  Meets	  requirements	  ENCODE	  Ab	  database:	  Eligible	  for	  use	  February 2014ENCODE	  Ab	  database:	  	  Not	  eligible	  for	  use	  6Use	  of	  a	  previously	  characterized	  lot	  number	  of	  an	  an$body	  in	  a	  new	  cell	  type	  (for	  TFs,	  chroma$n	  remodelers,	  chroma$n	  modiﬁers,	  RBPs)	  A	  Western	  blot	  or	  IP-­‐Western	  must	  be	  done	  using	  extracts	  from	  the	  new	  cell	  type	  Banding	  paTern	  is	  diﬀerent	  than	  in	  the	  cells	  for	  which	  that	  lot	  number	  of	  an5body	  has	  passed	  valida5on	  	  Banding	  paTern	  is	  the	  same	  as	  in	  the	  cells	  for	  which	  that	  lot	  number	  of	  an5body	  has	  passed	  valida5on	  	  Secondary	  characteriza5on	  is	  required:	  siRNA,	  tagging,	  ChIP-­‐seq#,	  Mass	  Spec*	  	  Does	  not	  meet	  Meets	  requirements	  	  requirements	  ENCODE	  Ab	  database:	  	  ENCODE	  Ab	  database:	  Not	  eligible	  for	  use	  Eligible	  for	  use	  No	  further	  characteriza5on	  is	  needed	  #not	  appropriate	  for	  RBP	  *not	  yet	  approved	  Antibody Characterization, ENCODE 3February 2014

7Use	  of	  a	  new	  lot	  #	  of	  a	  previously	  characterized	  an$body	  (for	  TFs,	  chroma$n	  remodelers,	  chroma$n	  modiﬁers)	  Western	  blot	  or	  IP-­‐Western	  of	  the	  new	  lot	  number	  using	  extracts	  from	  the	  same	  cell	  type	  used	  for	  the	  ChIP-­‐seq	  PaTern	  is	  the	  same	  as	  for	  the	  previously	  approved	  lot	  number	  PaTern	  is	  diﬀerent,	  but	  within	  ENCODE	  standards	  Meets	  requirements	  Secondary	  characteriza5on:	  siRNA,	  tagging,	  ChIP-­‐seq#,	  Mass	  Spec*	  wrong	  size,	  ladder	  of	  bands,	  no	  signal	  Does	  not	  meet	  requirements	  Meets	  requirements	  #not	  appropriate	  for	  RBP	  *not	  yet	  approved	  Antibody Characterization, ENCODE 3ENCODE	  Ab	  database:	  Eligible	  for	  use	  February 2014ENCODE	  Ab	  database:	  Not	  eligible	  for	  use	  8Characteriza$on	  of	  a	  modiﬁed	  histone	  an$body	  A	  Western	  blot	  or	  IP-­‐Western	  must	  be	  done	  for	  all	  lot	  numbers	  of	  any	  an5body	  extracts	  from	  at	  least	  3	  cell	  types	  should	  show	  signals	  only	  for	  histones	  AND	  recombinant	  unmodiﬁed	  histone	  protein	  should	  not	  be	  detected	  	  Meets	  requirements	  Pep5de	  arrays	  or	  pep5de	  compe55ons	  must	  show	  appropriate	  speciﬁcity	  Meets	  requirements	  ENCODE	  Ab	  database:	  Eligible	  for	  use	  in	  all	  cells	  for	  that	  species	  Antibody Characterization, ENCODE 3February 2014Cross	  reac5vity	  Does	  not	  meet	  requirements	  ENCODE	  Ab	  database:	  Not	  eligible	  for	  use	  9