Antibody validation criteria

We ask that user validations of Antibodypedia's inventory of antibodies in specific applications be accompanied by data and experimental details to facilitate the review process and community interpretation of results. Details of the antigen of interest, including any relevant post-translational modifications; the experimental setup (protocols or links to published methods); any primary or secondary antibodies used; and positive and negative controls will be requested during data submission. More specific guidelines can be found in the ‘Help’ boxes within the Antibodypedia manuscript tracking system. Please note that publication-quality data are not necessary, especially where results do not support antibody efficacy.


Gel shift
Flow cytometry
Chromatin IP/ RNA IP
Western blot
Immunoprecipitation
Microscopy (immunofluorescence, immunohistochemistry, immunocytochemistry, immunoelectron microscopy)
Blocking/neutralizing
ELISA/ELISPOT
Radioimmunoassay
Protein array

Gel shift

Supportive
  • A distinct supershifted band which recapitulates findings from an independent antibody against the same target protein appears when the antibody of interest is added to an antigen-oligonucleotide complex. Gel shift does not occur in the absence of antigen and/or with mutant antigen, scrambled or unlabelled probe or other negative control. Supershift does not occur with a nonspecific (isotype-matched) antibody or mock-/ pre-immune serum
  • A distinct supershifted band which recapitulates findings from an independent antibody against the same target protein appears when the antibody of interest is added to an antigen-oligonucleotide complex
  • Gel shift does not occur in the absence of antigen and/or with mutant antigen, scrambled or unlabelled probe or other negative control. Supershift occurs in the presence of the antibody of interest, but not with a nonspecific (isotype-matched) antibody or mock-/ pre-immune serum
  • Other
Nonsupportive
  • No supershift is detected upon antibody addition to an antigen-oligonucleotide complex despite a visible antigen-oligo band and verified antigen expression in the experimental sample
  • Incubation with the antibody does not result in discrete supershifting of the antigen-oligonucleotide complex
  • Results contradict previous observations with independently validated antibodies
  • Other

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Flow cytometry

Supportive
  • Incubation of a cell population with the antibody of interest results in a distinct increase in fluorescence matching that seen with an independent antibody against the antigen. Fluorescence does not increase when cells are incubated with a specific antibody and its competing peptide or with nonspecific (isotype-matched) antibody (or in the absence of antigen)
  • A fluorescence increase occurs when cells are incubated with the antibody of interest, but not during incubation with a specific antibody and its competing peptide or with nonspecific (isotype-matched) antibody (or in the absence of antigen)
  • Addition of the antibody of interest to a cell population results in a distinct increase in fluorescence matching that seen with an independent antibody against the antigen
  • Addition of the antibody of interest to a cell population results in a distinct increase in fluorescence only when cells are permeabilized (intracellular target)
  • Addition of the antibody of interest to a cell population results in a distinct increase in fluorescence only when cells express the target antigen (upon antigen induction or relative to a negative control sample with the antigen gene deleted or knocked down)
  • Other
Nonsupportive
  • Sample fluorescence does not increase upon addition of the antibody of interest despite independent verification of antigen expression
  • Fluorescence increases upon addition of the antibody of interest in a cell-type lacking antigen expression
  • Fluorescence increases upon addition of an antigen-specific antibody but does not recapitulate previous observations with an independent antibody (for instance, sorting of a cell population on a novel [isotype-matched] antibody and a validated antibody against an independent target generates an atypical scatter plot)
  • The antigen is intracellular but fluorescence increases upon antibody addition irrespective of cell permeabilization
  • Competition with a control peptide fails to inhibit an increase in fluorescence upon antibody addition
  • Other

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Chromatin IP/ RNA IP

Supportive
  • Antigen expression is known or predicted to occur in the cell-type assayed. An antibody against a nucleic-acid-binding protein immunoprecipitates the same nucleic-acid elements as an independent, previously validated antibody against the same target or its known interacting partner, as demonstrated by PCRs with verified primer sets. These nucleic-acid elements are not immunoprecipitated with mock-/ pre-immune serum or a control (isotype-matched) nonspecific antibody (or in an antigen-depleted sample)
  • Antigen expression is known or predicted to occur in the cell-type assayed. An antibody against a nucleic-acid-binding protein is assayed and validated prior to use by competition with a control peptide, as demonstrated by PCRs with verified primer sets. These nucleic-acid elements are not immunoprecipitated with mock-/ pre-immune serum or a control (isotype-matched) nonspecific antibody (or in an antigen-depleted sample)
  • No specific detection of antigen-bound nucleic-acid motifs is seen with mock-/ pre-immune serum, control nonspecific antibody or in a sample lacking antigen, as demonstrated by PCRs with verified primer sets
  • Other
Nonsupportive
  • An antibody against a verified nucleic-acid-binding protein does not immunoprecipitate, or nonspecifically immunoprecipitates, nucleic acid, as demonstrated by PCRs with control primer sets
  • The motifs immunoprecipitated are inconsistent with previous nucleic-acid-binding data for the antigen or its known interacting partner(s)
  • Control PCRs fail despite use of validated primers
  • Competition of the antigen-antibody complex with a control peptide fails; the antigen-antibody interaction is nonspecific
  • Other

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Western blot

Supportive
  • The antibody’s staining pattern is similar or identical to that of an independently validated positive-control antibody against the same target
  • The antibody’s staining pattern is similar or identical to that of an independently validated positive-control antibody against the same target. No antigen signal is detected with mock- or pre-immune serum/ control (isotype-matched) nonspecific antibody (or in tissue lacking antigen expression)
  • Band(s) recognized by antibody is (are) within 20% of the known or predicted antigen molecular weight. Additional bands are absent or faint
  • Band(s) recognized by antibody is (are) within 20% of the known or predicted antigen molecular weight. Additional bands are absent or faint. No signal is detected with mock- or pre-immune serum/ control (isotype-matched) nonspecific antibody (or in tissue known not to express antigen)
  • Band(s) recognized by the antibody is (are) within 20% of the known or predicted antigen molecular weight. Additional bands detected are consistent with a posttranslational modification known to occur in the tissue-type assayed or correspond to those observed with an independent, modification-specific, antibody
  • Band(s) recognized by the antibody is (are) within 20% of the known or predicted antigen molecular weight. Additional bands detected are consistent with a posttranslational modification known to occur in the sample assayed or correspond to those observed with an independent, modification-specific, antibody. No signal is detected with mock- or pre-immune serum/ control (isotype-matched) nonspecific antibody (or in tissue known not to express antigen)
  • Other
Nonsupportive
  • Antigen signal is not detected despite experimentally verified antigen expression in the tissue-type assayed
  • Antibody gives poor signal/ high background despite experimentally verified antigen expression in the sample assayed (including strong recognition of bands of incorrect size or lack of discrete bands)
  • Bands recognized by the antibody are inconsistent with the antigen’s molecular weight (discrepancy of more than 20%) despite an absence of antigen modification in the sample assayed
  • Bands recognized by the antibody are inconsistent with the antigen’s molecular weight (discrepancy of more than 20%) despite known antigen modification in the sample assayed
  • Other

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Immunoprecipitation

Supportive
  • Immunoprecipitation with the antibody of interest matches results from an independent positive-control antibody. Immunoprecipitation does not occur with non-immune or pre-immune serum
  • Immunoprecipitation with the antibody of interest, but not negative-control antibodies (mock- or pre-immune serum; control, isotype-matched nonspecific Ab) results in immunoprecipitation of the antigen and/ or co-immunoprecipitation of known or predicted antigen binding partners
  • Immunoprecipitation with the antibody of interest detects protein-protein interactions absent with a negative-control antibody or non-immune/ pre-immune serum (for uncharacterized protein complexes)
  • Immunoprecipitation with the antibody of interest detects protein-protein interactions known or predicted to occur in the sample-type assayed antigen-dependently (for instance, relative to knockdown, deletion, untransfected or uninduced controls)
  • Immunoprecipitation with the antibody of interest detects a unique set of protein-protein interactions antigen-dependently (for instance, relative to knockdown, deletion, untransfected or uninduced controls; for uncharacterized protein complexes)
  • Other
Nonsupportive
  • The antibody of interest fails to immunoprecipitate the antigen or its binding partners in tissues with verified antigen expression
  • The antibody of interest immunoprecipitates nonspecific proteins
  • Other

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Microscopy (immunofluorescence, immunohistochemistry, immunocytochemistry, immunoelectron microscopy)

Supportive
  • The antibody detects antigen in one or several compartments, as demonstrated by costaining, counterstaining or light microscopy. The staining pattern matches that seen with an independently validated antibody. No or diffuse staining is detected with a nonspecific (isotype-matched) antibody or mock-/ pre-immune serum (or in cells not expressing antigen: knockdown, deletion, untransfected or uninduced control)
  • The antibody detects antigen in one or several compartments, consistent with results from an independently validated antibody, as demonstrated by costaining, counterstaining or light microscopy
  • The antibody detects antigen in one or several compartments consistent with the literature or predictions, as demonstrated by costaining, counterstaining or light microscopy. No or diffuse staining is detected with a nonspecific (isotype-matched) antibody or mock-/ pre-immune serum (or in cells not expressing antigen: knockdown, deletion, untransfected or uninduced control)
  • The antigen localizes to one or several compartments consistent with the literature or predictions, as demonstrated by costaining, counterstaining or light microscopy
  • Other
Uncertain
  • The antibody localizes the antigen to multiple intracellular compartments, but previous studies suggested a single location
  • The antibody localizes the antigen to a single compartment, but previous studies suggested multiple locations
  • The antibody recognizes distinct antigen puncta, but signal partially overlaps or conflicts with previous (poorly substantiated) data
  • The antibody recognizes discrete antigen puncta, but signal partially overlaps or conflicts with previous data for the antigen’s validated binding partner
  • Other
Nonsupportive
  • Antibody staining results in no signal or diffuse/ nonspecific staining
  • Antigen localization by the antibody of interest is inconsistent with previous well substantiated data
  • Other

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Blocking/neutralizing

Supportive
  • The antibody of interest, but not a negative-control antibody or serum sample, recapitulates results for an independent neutralizing antibody or positive-control neutralizing serum against an antigen required for pathogen entry into the sample cell-type
  • The antibody recapitulates results for an independent neutralizing antibody or positive-control neutralizing serum against an antigen required for pathogen entry into the sample cell-type
  • The antibody, but not a negative-control antibody or serum sample, inhibits pathogen entry into a sample cell-type expressing its cognate antigen
  • Other
Nonsupportive
  • The antibody does not reduce or inhibit pathogen entry into the sample cell-type
  • Other

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ELISA/ELISPOT

Supportive
  • Antibody binding recapitulates that by an independent high-affinity antibody validated for ELISA, but not a nonspecific (isotype-matched) antibody. Assay results are within linear range of the standard curve
  • The affinity of the antigen-antibody interaction can be calibrated based on the presence of low- and high-affinity control antibodies, as well as a nonspecific (isotype-matched) control antibody. Assay results are within linear range of the standard curve
  • The affinity of the antigen-antibody interaction can be calibrated based on the presence of low- and high-affinity control antibodies. Assay results are within linear range of the standard curve
  • Other
Nonsupportive
  • The antibody of interest does not bind antigen
  • Antibody binding evinces high background/ nonspecific signal
  • Antigen binding by the antibody is similar to that of a low-affinity control antibody
  • Other

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Radioimmunoassay

Supportive
  • The antibody of interest detects antigen in a complex sample; antibody affinity for the antigen recapitulates that for an independent positive-control antibody, but not a nonspecific antibody. Assay results are within linear range of the standard curve
  • The affinity of the antigen-antibody interaction can be calibrated based on the presence of low- and high-affinity control antibodies, as well as a nonspecific control antibody. Assay results are within linear range of the standard curve
  • The affinity of the antigen-antibody interaction can be calibrated based on the presence of low- and high-affinity control antibodies. Assay results are within linear range of the standard curve
  • Other
Nonsupportive
  • The antibody does not bind antigen
  • Antibody binding evinces high background/ nonspecific signal
  • Antigen binding by the antibody is similar to that of a low-affinity control antibody
  • Other

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Protein array

Supportive
  • The antibody binds exclusively to its cognate antigen (antigen array)
  • An antigen binds exclusively to the antibody of interest (antibody array)
  • Other
Nonsupportive
  • Antigen-antibody interactions are nonspecific (interactions between the antibody and an additional single antigen constitute at least 40% of signal or interactions between the antibody and two or more additional antigens comprise at least 15% [each] of signal)
  • Antigen-antibody interactions are weak or absent (low signal/ high background)
  • Other