Atlas Antibodies
The goal of Atlas Antibodies is to provide customers with advanced research reagents targeting all human proteins. In close partnership with the Human Protein Atlas (HPA) project, they produce unique, highly characterised antibodies. Their product range consists of over 17,000 antibodies covering 15,000 gene products and an additional 13,000 protein targets.
Cambridge Bioscience is a distributor of Atlas Antibodies in the UK and Ireland.
Atlas’s antibodies are rigorously evaluated for specificity and performance. An additional level of antibody validation (‘Enhanced Validation’) is being added following the recommendations by the International Working Group for Antibody Validation (IWGAV) published in Nature Methods. Over 4,200 antibodies from Atlas’s catalogue have undergone this enhanced validation. They have proposed five conceptual pillars for antibody validation, which should be used in an application-specific manner. At least one of the pillars should be used for an antibody to be validated in a specific application. Atlas Antibodies base the enhanced validation on Human Protein Atlas' interpretation of these pillars.
Genetic Validation
• Downregulate the target protein on a genetic level using siRNA or CRISPR-Cas9
• Antibody specificity confirmed when knockout or knockdown of the corresponding gene correlates with absence or decrease of the antibody signal
• Used at Atlas Antibodies to validate antibodies in WB
• Method can also be applied for antibody validation in ICC-IF
Downregulation of antibody signal confirming target specificity. Western blot analysis of extracts from U-251 cells, transfected with either control siRNA (siRNA ctrl) or target specific siRNA probes (siRNA#1, siRNA#2), using Anti-PPIB monoclonal antibody (AMAb91245). Remaining relative intensity after silencing is indicated.
Orthogonal Validation
• Compare results with a non-antibody based method across multiple samples e.g. antibody staining intensities to RNA-Seq data from the same samples, over multiple tissues or cells with varying expression of the target protein
• Antibody specificity confirmed when antibody signal matches RNA levels in evaluated samples
• For each antibody, 2 tissues/endogenous cell lines chosen for validation, one with high RNA expression & the other with low or no RNA expression of the target
• Comparing antibody signal to RNA-Seq data is used at Atlas Antibodies to validate antibodies in WB & IHC
• Method can also be applied for ICC-IF
Examples of orthogonal validation in WB and IHC. Left: WB analysis in human cell lines SK-MEL-30 and Caco-2 using Anti-RAB27A antibody (HPA001333). Corresponding RAB27A RNA-seq data (TPM values) is presented for the same cell lines. Right: IHC staining of liver and colon tissues using the Anti- SLC2A2 antibody (HPA028997). The corresponding RNA-Seq data (TPM values) for the same tissues are presented below. In both examples, samples with known high and low RNA expression are chosen.
Validation by Independent Antibodies
• Compare 2 antibodies targeting different regions of the same protein
• If the two antibodies generate a similar staining pattern when compared in a set of relevant tissues, the antibodies validate each other
• Independent antibody validation can be applied in IHC, WB & ICC-IF setups
• Approach requires:
- Exact knowledge of the antigen sequence for each antibody
- At least two antibodies with non-overlapping antigen sequences for each protein target
The two Anti-A2ML1 antibodies HPA038847 (left) and HPA038848 (right) target different regions of A2ML1. Antibody stainings across relevant positive and negative tissues are similar between the two, and the antibodies validate each other’s staining pattern in IHC. Here, each of the two antibodies are stained using esophagus (positive for A2ML1 using both antibodies), tonsil, colon and kidney (negative for A2ML1 using both antibodies).
Recombinant Expression Validation
• Antibody binding verified using an over-expressed or a 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
• Approach can be applied in WB
Example of recombinant expression validation in Western blot using the Anti-ACY3 antibody (HPA039219). Lane 1: marker, lane 2: negative control (vector only transfected HEK293T lysate), lane 3: ACY3 over-expression lysate (Co-expressed with a C-terminal myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells, LY408962).
Migration Capture Mass Spectrometry (MS) Validation
• Staining pattern & protein size detected by the antibody is compared with results obtained by capture MS
• Antibody specificity is confirmed when the size detected by the antibody is equivalent to the size of the corresponding target protein detected in migration capture MS
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