AHNAK - Antibodies - The Human Protein Atlas


	Antibody HPA019010	Antibody HPA019070	Antibody HPA026643

ANTIBODY INFORMATION
Provider	Atlas Antibodies Sigma-Aldrich	Atlas Antibodies Sigma-Aldrich	Atlas Antibodies Sigma-Aldrich
Product name	HPA019010	HPA019070	HPA026643
Host species	Rabbit	Rabbit	Rabbit
Clonalityⁱ The antibodies are designated mAB for monoclonal and pAb for polyclonal.	pAb	pAb	pAb
Concentration	0.0175 mg/ml	0.11 mg/ml	0.0625 mg/ml
Purity	Affinity purified using the PrEST-antigen as affinity ligand	Affinity purified using the PrEST-antigen as affinity ligand	Affinity purified using the PrEST-antigen as affinity ligand
Released in versionⁱ The release of the Human Protein Atlas in which the antibody was first published.	4.1	4.1	7.0
Referencesⁱ References to publications in which the antibody has been used.	1	2	2
Proper citation	Atlas Antibodies Cat#HPA019010, RRID:AB_1844657	Atlas Antibodies Cat#HPA019070, RRID:AB_1844656	Atlas Antibodies Cat#HPA026643, RRID:AB_10670426
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 N/A WB PA	ICC IHC N/A WB PA	ICC IHC N/A WB 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.	Enhanced - Independent antibodiesⁱ This method is based on comparing the staining pattern using two independent antibodies with no overlapping epitopes. A similar staining pattern (main and additional locations) results in an enhanced antibody validation. Antibody staining overlaps with antibody HPA019070. Immunofluorescent staining of human cell line U2OS shows localization to plasma membrane and cytosol. Protein location across all samples similar between independent antibodies	Enhanced - Independent antibodiesⁱ This method is based on comparing the staining pattern using two independent antibodies with no overlapping epitopes. A similar staining pattern (main and additional locations) results in an enhanced antibody validation. Antibody staining overlaps with antibody HPA019010. Immunofluorescent staining of human cell line A-431 shows localization to plasma membrane and cytosol. Protein location across all samples similar between independent antibodies	Uncertainⁱ 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 not consistent with literature. Immunofluorescent staining of human cell line U2OS shows localization to plasma membrane, vesicles and cytosol.

Antibody dilution	Human assay: A-431 fixed with PFA, dilution: 1:9 Human assay: U-251MG fixed with PFA, dilution: 1:9 Human assay: U2OS fixed with PFA, dilution: 1:9	Human assay: A-431 fixed with PFA, dilution: 1:56 Human assay: U-251MG fixed with PFA, dilution: 1:56 Human assay: U2OS fixed with PFA, dilution: 1:56	Human assay: A-431 fixed with PFA, dilution: 1:20 Human assay: U-251MG fixed with PFA, dilution: 1:20 Human assay: U2OS fixed with PFA, dilution: 1:20
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.	Uncertainⁱ Immunohistochemistry is used for validating antibody reliability by assessing staining pattern in 44 normal tissues. Validation scores include Enhanced, Supported, Approved and Uncertain. Immunohistochemical staining of human oral mucosa shows cytoplasmic positivity in squamous epithelial cells. Oral mucosa	Uncertainⁱ Immunohistochemistry is used for validating antibody reliability by assessing staining pattern in 44 normal tissues. Validation scores include Enhanced, Supported, Approved and Uncertain. Immunohistochemical staining of human rectum shows cytoplasmic positivity in glandular cells. Rectum	Uncertainⁱ Immunohistochemistry is used for validating antibody reliability by assessing staining pattern in 44 normal tissues. Validation scores include Enhanced, Supported, Approved and Uncertain. Immunohistochemical staining of human small intestine shows strong membranous and cytoplasmic positivity in glandular cells. Small intestine

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 pH6	HIER pH6
Antibody dilution	1:10	1:500	1:250
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.	Partly consistent with gene/protein characterization data.	Partly consistent with gene/protein characterization data.	Partly consistent with 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.	Low consistency between antibody staining and RNA expression data.	Low consistency between antibody staining and RNA expression data.	Low 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.	Uncertainⁱ 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. Target too small/large to be analyzed with the present setup. Analysis performed using a standard panel of samples.	Uncertainⁱ 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. Target too small/large to be analyzed with the present setup. Analysis performed using a standard panel of samples.	Uncertainⁱ 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. Target too small/large to be analyzed with the present setup. Analysis performed using a standard panel of samples.

Antibody dilution	1:250	1:250	1:250
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.	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.	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.

Antibody dilution	1:500	1:3000	1:3000
RELEVANT PUBLICATIONS
	Contribution of antibody-based protein profiling to the human Chromosome-centric Proteome Project (C-HPP) Fagerberg L et al J Proteome Res 2013;12(6):2439-48	Contribution of antibody-based protein profiling to the human Chromosome-centric Proteome Project (C-HPP) Fagerberg L et al J Proteome Res 2013;12(6):2439-48	AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility Silva TA et al Oncotarget 2016;7(31):49998-50016 Application: IHC
		AHNAK and inflammatory markers predict poor survival in laryngeal carcinoma Dumitru CA et al PLoS One 2013;8(2):e56420	Proteomic Signatures of Thymomas Wang L et al PLoS One 2016;11(11):e0166494 Application: IHC
ANTIGEN INFORMATION
Antigen	Recombinant protein fragment	Recombinant protein fragment	Recombinant protein fragment
Length (aa)	134	124	132
Antigen sequence	`LGEGHLSVKGSGGEWKGPQVSSALNLDTSKFAGGLHFSGPKVEGGVKGGQ IGLQAPGLSVSGPQGHLESGSGKVTFPKMKIPKFTFSGRELVGREMGVDV HFPKAEASIQAGAGDGEWEESEVKLKKSKIKMPK`	`DIKSPSLDVTVPEAELNLETPEISVGGKGKKSKFKMPKIHMSGPKIKAKK QGFDLNVPGGEIDASLKAPDVDVNIAGPDAALKVDVKSPKTKKTMFGKMY FPDVEFDIKSPKFKAEAPLPSPKL`	`MKGTKVKTPEMIIQKPKISMQDVDLSLGSPKLKGDIKVSAPGVQGDVKGP QVALKGSRVDIETPNLEGTLTGPRLGSPSGKTGTCRISMSEVDLNVAAPK VKGGVDVTLPRVEGKVKVPEVDVRGPKVDVSA`
Matching transcripts	AHNAK-202 - ENSP00000367263 [100%]	AHNAK-202 - ENSP00000367263 [100%]	AHNAK-202 - ENSP00000367263 [100%]
Matching mouse transcripts	ENSMUSP00000090633 [87%] ENSMUSP00000145598 [28%] ENSMUSP00000159404 [26%] ENSMUSP00000158470 [17%]	ENSMUSP00000090633 [86%] ENSMUSP00000159404 [27%] ENSMUSP00000135941 [24%]	ENSMUSP00000090633 [81%] ENSMUSP00000159404 [32%] ENSMUSP00000066110 [25%]
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.

AHNAK-202

Displaying protein features on the AlphaFold structures

The Protein Browser