Enhanced Validation of Specific Antibodies from Atlas Antibodies

Validation of antibodies is an experimental confirmation that an antibody is suitable for its intended use. It is important to understand that antibody validation is application- and context specific. Atlas Antibodies provides highly characterized antibodies further strengthened by enhanced validation, as recommended by leading researchers and described in this paper.

Triple A polyclonals

Triple A polyclonals are originally developed within the academic Human Protein Atlas (HPA) project (proteinatlas.org). HPA uses the antibodies to characterize all human proteins in the majority of normal and cancerous tissues, as well as on a subcellular level. The uniqueness and specificity of the antibodies used, originate from a thorough selection of antigen regions, affinity purification of the polyclonal antibodies, a stringent selection of approved antibodies and validation using different methods.

PrecisA monoclonals

Atlas Antibodies also provides a selected number of mouse monoclonal antibodies, under the brand name PrecisA monoclonals. PrecisA monoclonals are developed with careful antigen design and are isotyped for multiplex possibilities.

Transparency of data

  • All antibody characterization data achieved using different applications are freely accessible on the Human Protein Atlas portal.
  • For immunohistochemistry (IHC), every antibody is supplied with 500 images, from 44 normal and 20 cancer tissues.
  • The antigen sequences are provided on the product pages for all antibodies.
  • Precise epitope information is supplied for PrecisA monoclonals (where available).
  • The scientific support team will be happy to share anything you may like to know about production and quality control of each antibody.

Enhanced validation of antibodies

To further strenghten the reliability of their antibodies Atlas Antibodies apply enhanced validation, based on the recommendations by the International Working Group for Antibody Validation (IWGAV), as published in Nature Methods, Uhlén et al1. The group consists of researchers from institutions in USA and Canada, such as Stanford University, Yale University, MIT, UCSD, University of Toronto, NIH, EMBL, Niigata University in Japan and the Science for Life laboratory in Sweden. In the article, five conceptual pillars for antibody validation are suggested to be used in an application specific manner. Based on these pillars, HPA and Atlas Antibodies have developed an enhanced validation strategy, resulting in the following five methods:

Five conceptual methods

  1. Genetic validation: Target confirmed by siRNA knock-down.
  2. Orthogonal validation: Specificity confirmed by a non-antibody based method.
  3. Independent antibody validation: Specificity confirmed by another antibody targeting a different epitope of the protein.
  4. Recombinant expression validation: Terget confirmed by an overexpressed version of the protein.
  5. Capture MS validation: Presence of target verified by mass spectrometry.
At least one of the methods must be used for an antibody to receive enhanced validation status in a specific application.

1. Genetic validation

The target protein can be down-regulated on a genetic level using siRNA or CRISPR-Cas9. If knockdown (by siRNA) or knock-out (by CRISPR) of the corresponding gene correlates with absence or decrease of antibody signal, the antibody is shown to be specific for its target. This is exemplified in Figure 1 where the Anti-PPIB antibody AMAb91245 is used in WB analysis in U-251 cells. The antibody signal is down regulated in the cells silenced by PPIB siRNA.

2. Orthogonal validation

By using a non-antibody based method for target quantification, the antibody signal can be validated by comparing the results from the different methods across multiple samples. One approach is to compare the antibody staining intensities over multiple samples with varying expression of the target gene, with RNA-seq data in the same samples. This is illustrated in Figure 2, using WB analysis in human cell lines SK-MEL-30 and Caco-2 using Anti-RAB27A antibody HPA001333. Corresponding RAB27A RNA-seq data (TPM values) are presented for the same cell lines.

3. Validation by independent antibodies

Two antibodies directed against different regions of the same target protein may validate each other when compared in a set of relevant tissues. This is exemplified in Figure 3 with two antibodies directed against different regions of the A2ML1 protein used in the immunohistochemistry application. Both antibodies show positive staining in liver, but are negative in tonsil, colon and kidney.

4. Recombinant expression validation

An antibody signal can also be validated using an overexpressed or tagged version of the target protein. When over-expressing the target protein in a cell line, the antibody is validated by comparing the signal from the over-expressed version with the unmodified endogenous target protein. This approach is exemplified in Figure 4a.

The target protein may also be tagged by an affinity tag or a fluorescent protein. The pattern displayed by the tagged target protein is matched to the antibody signal.

A match confirms that the antibody recognizes its target protein. Validation by tagged proteins can be applied in immunocytochemistry-immunofluorescence (ICC-IF) and is shown in Figure 4b.

5. Migration capture MS validation

In this method, the staining pattern and the protein size detected by the antibody is compared with results obtained by a capture mass spectrometry method. The band generated by the antibody in WB should correlate with the target protein identified by MS in terms of gel migration.

Atlas Antibodies and validation

At Atlas Antibodies we offer highly characterized antibodies extensively validated in collaboration with the Human Protein Atlas, and implement enhanced validation providing an additional layer of security.

Enhanced validation guidelines

Antibodies with enhanced validation (selection from 7000+ products)

Cat. No.DescriptionApplicationsVerified Species Reactivity
HPA001352APOA4 Antibody, Rabbit PAbIF, IHCHuman
HPA018885APOL1 Antibody, Rabbit PAbIHC, WBHuman
HPA014784AQP4 Antibody, Rabbit PAbIHC, WBHuman, Mouse, Rat
AMAb90784ATRX Antibody, Mouse MAb, [CL0537]IHCHuman
HPA001906ATRX Antibody, Rabbit PAbIHCHuman
HPA045218BASP1 Antibody, Rabbit PAbIHCHuman
HPA023013BICD2 Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
HPA046744C19orf10 Antibody, Rabbit PAbIHC, WBHuman
HPA023778C1orf112 Antibody, Rabbit PAbIHC, WBHuman
HPA023195CALCOCO2 Antibody, Rabbit PAbIF, IHC, WBHuman
HPA024470CAPN2 Antibody, Rabbit PAbIHC, WBHuman, Mouse, Rat
HPA020626CCNA2 Antibody, Rabbit PAbIHC, WBHuman
AMAb90897CD14 Antibody, Mouse MAb, [CL1637]IHC, WBHuman
HPA004252CD4 Antibody, Rabbit PAbIHC, WBHuman
HPA017369CHGA Antibody, Rabbit PAbIHCHuman
HPA014704CMTM4 Antibody, Rabbit PAbIHC, WBHuman
HPA052963COL17A1 Antibody, Rabbit PAbIHCHuman
HPA029025CPSF3L Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
HPA013835CRB3 Antibody, Rabbit PAbIHCHuman
HPA051227CYBB Antibody, Rabbit PAbIHC, WBHuman
HPA008736DAXX Antibody, Rabbit PAbIF, IHC, WBHuman
HPA039715DCTN2 Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
HPA003010DLST Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
HPA054950DSP Antibody, Rabbit PAbIHCHuman
AMAb90562EMD Antibody, Mouse MAb, [CL0203]IF, IHC, WBHuman
HPA026761EPCAM Antibody, Rabbit PAbIHC, WBHuman
HPA021616EZR Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
HPA069613FOXL2 Antibody, Rabbit PAbICC-IF, IHCHuman
AMAb90549FUS Antibody, Mouse MAb, [CL0190]IF, IHC, WBHuman
HPA036359GCLC Antibody, Rabbit PAbIHC, WBHuman
HPA003418GZMB Antibody, Rabbit PAbIF, IHC, WBHuman
HPA002083HNF1B Antibody, Rabbit PAbIF, IHC, WBHuman
HPA037850HORMAD1 Antibody, Rabbit PAbIF, IHCHuman
AMAb91096LAMB2 Antibody, Mouse MAb, [CL2976]IHC, WBHuman
HPA051467LAMP3 Antibody, Rabbit PAbIHCHuman
HPA021942MAGEA4 Antibody, Rabbit PAbIHCHuman
HPA069392MLN Antibody, Rabbit PAbIHCHuman
HPA014144NECAB2 Antibody, Rabbit PAbIHCHuman
HPA003097NF2 Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
HPA014518P2RY12 Antibody, Rabbit PAbIHC, WBHuman
AMAb90956P53 Antibody, Mouse MAb, [CL2199]IHC, WBHuman
HPA052794PALM3 Antibody, Rabbit PAbIHCHuman
AMAb90803PGAM5 Antibody, Mouse MAb, [CL0624]IHC, WBHuman
HPA018798PIWIL1 Antibody, Rabbit PAbIHCHuman
AMAb90655RBM3 Antibody, Mouse MAb, [CL0296]IHC, WBHuman
AMAb90594RUNX2 Antibody, Mouse MAb, [CL0235]IHC, WBHuman
HPA007575S100A6 Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
AMAb90635SATB2 Antibody, Mouse MAb, [CL0276]IHC, WBHuman
AMAb90708SDHB Antibody, Mouse MAb, [CL0349]IHC, WBHuman
AMAb90777STAT3 Antibody, Mouse MAb, [CL0492]IHC, WBHuman
HPA001475SYTL4 Antibody, Rabbit PAbIF, IHC, WBHuman
HPA023881TFE3 Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
HPA029480TKT Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
HPA051870TMEM119 Antibody, Rabbit PAbIHCHuman
HPA003037TOMM22 Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse, Rat
AMAb90516VIM Antibody, Mouse MAb, [CL0157]IHC, WBHuman
HPA001762VIM Antibody, Rabbit PAbIF, IHC, WBHuman, Mouse
HPA003041VPREB3 Antibody, Rabbit PAbIF, IHC, WBHuman
AMAb90730WWTR1 Antibody, Mouse MAb, [CL0371]IHC, WBHuman
HPA027524ZEB1 Antibody, Rabbit PAbIF, IHC, WBHuman
HPA003456ZEB2 Antibody, Rabbit PAbIF, IHCHuman

Related to:
Brands: Atlas Antibodies
Product groups: Antibodies