VIM - Antibodies - The Human Protein Atlas


	Antibody HPA001762	Antibody CAB000080	Antibody CAB058687

ANTIBODY INFORMATION
Provider	Atlas Antibodies Sigma-Aldrich	Agilent (Formerly DakoCytomation)	Atlas Antibodies
Product name	HPA001762	M7020	AMAb90516
Host species	Rabbit	Mouse	Mouse
Clonalityⁱ The antibodies are designated mAB for monoclonal and pAb for polyclonal.	pAb	mAb	mAb
Concentration	0.15 mg/ml	Not known	Not known
Purity	Affinity purified using the PrEST-antigen as affinity ligand	Protein A/G	Protein A/G
Released in versionⁱ The release of the Human Protein Atlas in which the antibody was first published.	2.0	1.2	11.0
Referencesⁱ References to publications in which the antibody has been used.	11
Proper citation	Atlas Antibodies Cat#HPA001762, RRID:AB_1080568	Agilent Cat#M7020, RRID:AB_2304493	Atlas Antibodies Cat#AMAb90516, RRID:AB_2665570
Validation summaryⁱ All assays through which the antibody has been validated. Assays&annotation provide a detailed description of the different assays. The pie-charts indicate degree of validation.	ICC IHC WB PA	N/A ICC IHC WB N/A PA	N/A ICC IHC WB N/A PA
IMMUNOCYTOCHEMISTRYⁱ Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.
Validationⁱ Results of validation by standard or enhanced validation. Standard validation is based on concordance with available experimental gene/protein characterization data in the UniProtKB/Swiss-Prot database. Standard validation results in scores Supported, Approved or Uncertain. Enhanced validation is performed using either siRNA knockdown, tagged GFP cell lines or independent antibodies. For the siRNA validation the decrease in antibody-based staining intensity upon target protein downregulation is evaluated. For the GFP validation the signal overlap between the antibody staining and the GFP-tagged protein is evaluated. For the independent antibodies validation the evaluation is based on comparison of the staining of two (or more) independent antibodies directed towards independent epitopes on the protein. For all cases except the siRNA validation, an image representative of the antibody staining pattern is shown. For the siRNA validation, a box plot of the results is shown.	Supportedⁱ Reliability scores for antibodies used in immunocytochemistry are set by comparing the staining pattern in cell lines with external experimental evidence for protein localization. The scores are termed Supported, Approved and Uncertain. The subcellular location is supported by literature. Immunofluorescent staining of human cell line ASC52telo shows localization to intermediate filaments.	N/A	N/A

Antibody dilution	Human assay: ASC52telo fixed with PFA, dilution: 1:75 Human assay: U-251MG fixed with PFA, dilution: 1:74 Human assay: U2OS fixed with PFA, dilution: 1:74
IMMUNOHISTOCHEMISTRYⁱ Immunohistochemistry is used for validating antibody reliability by assessing staining pattern in 44 normal tissues. Validation scores include Enhanced, Supported, Approved and Uncertain.
Validationⁱ Results of validation by standard or enhanced validation based on assessment of antibody performance in 44 normal tissues. Standard validation results in scores Supported, Approved or Uncertain. An image representative of the antibody staining pattern is shown. Enhanced validation results in the score Enhanced and includes two methods: Orthogonal validation and Independent antibody validation. For orthogonal validation, representative images of high and low expression are shown. For independent antibody validation, four images of each independent antibody are displayed.	Enhanced - Orthogonal Antibody staining mainly consistent with RNA expression data across 44 tissues. HIGH EXPRESSION Ovary RNA expression: 3594.6 nTPM LOW EXPRESSION Skeletal muscle RNA expression: 351.1 nTPM	Enhanced - Orthogonal Antibody staining mainly consistent with RNA expression data across 44 tissues. HIGH EXPRESSION Ovary RNA expression: 3594.6 nTPM LOW EXPRESSION Skeletal muscle RNA expression: 351.1 nTPM	Enhanced - Orthogonal Antibody staining mainly consistent with RNA expression data across 44 tissues. HIGH EXPRESSION Ovary RNA expression: 3594.6 nTPM LOW EXPRESSION Skeletal muscle RNA expression: 351.1 nTPM

Retrievalⁱ Antigen retrieval is a method used to restore/retrieve the epitope (antibody bidning region) of the target protein, cross-linked, and thus masked, during tissue preserving fixative treatment of the tissues.	HIER pH6	HIER pH9	HIER pH6
Antibody dilution	1:1000	1:400	1:5000
Literature conformityⁱ Conformance of the expression pattern with available gene/protein characterization data in scientific literature and data from bioinformatic predictions. UniProt is used as the main source of gene/protein characterization data and when relevant, available publications and other sources of information are researched in depth. Extensive or sufficient gene/protein data requires that there is evidence of existence on a protein level and that a substantial quantity of published experimental data is available from literature and public databases. Limited protein/gene characterization data does not require evidence of existence on a protein level and refers to genes for which only bioinformatic predictions and scarce published experimental data is available.	Consistent with extensive gene/protein characterization data.	Consistent with extensive gene/protein characterization data.	Consistent with extensive gene/protein characterization data.
RNA consistencyⁱ Consistency between immunohistochemistry data and consensus RNA levels is divided into five different categories: i) High consistency, ii) Medium consistency, iii) Low consistency, iv) Very low consistency, and v) Cannot be evaluated.	High consistency between antibody staining and RNA expression data.	High consistency between antibody staining and RNA expression data.	High consistency between antibody staining and RNA expression data.
WESTERN BLOTⁱ A Western blot analysis is performed on a panel of human tissues and cell lines to evaluate antibody specificity. For antibodies with unreliable result a revalidation using an over-expression lysate is performed.
Validationⁱ Western Blot is used for quality control of the polyclonal antibodies generated in the project. After purification, the antibodies are used to detect bands in a setup of lysate and different tissues. The result is then scored Enhanced, Supported, Approved, or Uncertain. Enhanced validation includes five different methods: Genetic validation, Recombinant expression validation, Independent antibody validation, Orthogonal validation and Capture MS validation.	Enhanced - Orthogonalⁱ This method is based on manual evaluation by comparing the antibody band intensity against the corresponding protein levels quantified by mass spectrometry (MS) or expression determined by RNA-seq. Antibodies are considered enhanced where the staining intensity and protein expression levels show the same expression pattern. A standard panel of two cell lines (RT4 and U-251) are used and the target protein must express the target at different levels. Antibody band intensities is confirmed by MS TMT data. 206 113 82 49 32 26 18	Supportedⁱ The staining of an antibody is evaluated by Western Blot through analysis of samples from different cell lysates. A supportive score is given if band(s) of predicted size in kDa (+/-20%) is detected. Band of predicted size in kDa (+/-20%) with additional bands present. Analysis performed using a standard panel of samples. 230 110 82 49 32 26 18	Supportedⁱ The staining of an antibody is evaluated by Western Blot through analysis of samples from different cell lysates. A supportive score is given if band(s) of predicted size in kDa (+/-20%) is detected. Band of predicted size in kDa (+/-20%) with additional bands present. Analysis performed using a standard panel of samples. 250 130 100 70 55 35 25 15 10

Antibody dilution	1:250	1:750	1:500
PROTEIN ARRAY
Validationⁱ A protein array containing 384 different antigens including the antibody target is used to analyse antibody specificity. Depending on the array interaction profile the antibody is scored as Supported, Approved, or Uncertain.	Supported Pass with single peak corresponding to interaction only with its own antigen. Antibody specificity analysis with protein arrays. Predicted and matching interactions are shown in green.	N/A	N/A

Antibody dilution	1:3000
RELEVANT PUBLICATIONS
	Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy Stadler C et al J Proteomics 2012;75(7):2236-51
	Tumor suppressive microRNAâ€‘138 contributes to cell migration and invasion through its targeting of vimentin in renal cell carcinoma Yamasaki T et al Int J Oncol 2012;41(3):805-17
	Regulation of glutamine carrier proteins by RNF5 determines breast cancer response to ER stress-inducing chemotherapies Jeon YJ et al Cancer Cell 2015;27(3):354-69 Application: IHC
	Changes in cannabinoid receptors, aquaporin 4 and vimentin expression after traumatic brain injury in adolescent male mice. Association with edema and neurological deficit Lopez-Rodriguez AB et al PLoS One 2015;10(6):e0128782 Application: WB
	Inhibition of Lysyl Oxidase and Lysyl Oxidase-Like Enzymes Has Tumour-Promoting and Tumour-Suppressing Roles in Experimental Prostate Cancer Nilsson M et al Sci Rep 2016;6:19608 Application: IHC
	Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression Sawant S et al PLoS One 2016;11(8):e0160615 Application: ICC-IF
	Extracellular Vesicles from Metastatic Rat Prostate Tumors Prime the Normal Prostate Tissue to Facilitate Tumor Growth Halin Bergström S et al Sci Rep 2016;6:31805 Application: IHC
	A 3D in vitro model of the human breast duct: a method to unravel myoepithelial-luminal interactions in the progression of breast cancer Carter EP et al Breast Cancer Res 2017;19(1):50 Application: ICC-IF, IHC
	2-Arachidonoylglycerol Reduces Proteoglycans and Enhances Remyelination in a Progressive Model of Demyelination Feliú A et al J Neurosci 2017;37(35):8385-8398 Application: ICC-IF
	Development of a predictive model for stromal content in prostate cancer samples to improve signature performance Boufaied N et al J Pathol 2019;249(4):411-424 Application: IHC
	Ginkgolic Acid, a SUMO-1 Inhibitor, Inhibits the Progression of Oral Squamous Cell Carcinoma by Alleviating SUMOylation of SMAD4 Liu K et al Mol Ther Oncolytics 2020;16:86-99 Application: ICC-IF, WB
ANTIGEN INFORMATION
Antigen	Recombinant protein fragment	Native protein	Recombinant protein
Length (aa)	113
Antigen sequence	`FANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQ LTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNA SLARLDLERKVES`
Matching transcripts	VIM-201 - ENSP00000224237 [100%] VIM-209 - ENSP00000446007 [100%]
Matching mouse transcripts	ENSMUSP00000028062 [99%] ENSMUSP00000141494 [99%] ENSMUSP00000027409 [67%] ENSMUSP00000077061 [61%] ENSMUSP00000114742 [19%]
ANTIGEN VIEWⁱ The Structure section provides in-house generated structures, predicted using the Alphafold source code, for the majority of the proteins and their related isoforms. Displaying protein features on the AlphaFold structures Individual splice variants can be selected in the top part of the Protein Browser (see below) and different transcript-related features such as transmembrane regions, InterPro domains and antigen sequences for antibodies can be displayed in the structure by clicking on the respective features in the Protein Browser. Clinical and population-based amino acid variants based on data from the Ensembl variation database and AlphaMissense (AM) predictions can be highlighted using the sliders to the right of the structure. These can also be used to colour the entire structure by residue index or make the structure autorotate.The structures are displayed using the NGL Viewer and can also be zoomed-in and rotated manually. The Protein Browser The ProteinBrowser displays the antigen location on the target protein(s) and the features of the target protein. Transcript names and schematic transcript structures including exons, introns and UTRs for the different isoforms are shown on top, and can be used to switch between the structures for the different splice variants. At the top of the view, the position of the antigen (identified by the corresponding HPA identifier) is shown as a green bar. A yellow triangle on the bar indicates a <100% sequence identity to the protein target. Below the antigens, the maximum percent sequence identity of the protein to all other proteins from other human genes is displayed, using a sliding window of 10 aa residues (HsID 10) or 50 aa residues (HsID 50). The region with the lowest possible identity is always selected for antigen design, with a maximum identity of 60% allowed for designing a single-target antigen (read more). The curve in blue displays the predicted antigenicity i.e. the tendency for different regions of the protein to generate an immune response, with peak regions being predicted to be more antigenic.The curve shows average values based on a sliding window approach using an in-house propensity scale. (read more). Signal peptides (turquoise) and membrane regions (orange) based on predictions using the majority decision methods MDM and MDSEC are also displayed. Low complexity regions are shown in yellow and InterPro regions in green. Common (purple) and unique (grey) regions between different splice variants of the gene are also displayed (read more), and at the bottom of the protein view is the protein scale.

VIM-201 VIM-209

Displaying protein features on the AlphaFold structures

The Protein Browser