DAXX - Antibodies - The Human Protein Atlas


	Antibody HPA008736	Antibody HPA008797	Antibody HPA065779	Antibody CAB002224	Antibody CAB025546

ANTIBODY INFORMATION
Provider	Atlas Antibodies Sigma-Aldrich	Atlas Antibodies Sigma-Aldrich	Atlas Antibodies Sigma-Aldrich	Leica Biosystems (Formerly Novocastra)	R&D Systems
Product name	HPA008736	HPA008797	HPA065779	NCL-DAXX	AF4548
Host species	Rabbit	Rabbit	Rabbit	Mouse	Goat
Clonalityⁱ The antibodies are designated mAB for monoclonal and pAb for polyclonal.	pAb	pAb	pAb	mAb	msAb
Concentration	0.0375 mg/ml	0.0475 mg/ml	0.2495 mg/ml	Not known	Not known
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	Not known	Affinity
Released in versionⁱ The release of the Human Protein Atlas in which the antibody was first published.	3.1	12.0	14.0	1.2	5.0
Referencesⁱ References to publications in which the antibody has been used.	22
Proper citation	Atlas Antibodies Cat#HPA008736, RRID:AB_1078625	Atlas Antibodies Cat#HPA008797, RRID:AB_2797218	Atlas Antibodies Cat#HPA065779, RRID:AB_2685558	Leica Biosystems Cat#NCL-DAXX, RRID:AB_563688	n/a
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.	N/A ICC IHC WB PA	ICC N/A IHC WB PA	ICC N/A IHC WB PA	N/A ICC IHC N/A WB N/A PA	N/A ICC IHC N/A 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.	N/A	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 HPA065779. Immunofluorescent staining of human cell line A-431 shows localization to nucleoplasm and nuclear bodies. Protein location across all samples similar between independent antibodies Enhanced - Recombinant expressionⁱ Antibody validation by comparing the staining pattern of the antibody targeting the endogenous protein with that of a GFP-tagged recombinant version of the protein. Enhance validation is given when antibody staining of the endogenous protein overlaps with the tagged protein, potentially also with some additional localizations. Antibody staining overlaps with GFP tagged protein Immunofluorescent staining of transgenic HeLa cells show antibody staining in nucleoplasm & nuclear bodies and GFP expression in nucleoplasm & nuclear bodies.	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 HPA008797. Immunofluorescent staining of human cell line SH-SY5Y shows localization to nucleoplasm and nuclear bodies. Protein location across all samples similar between independent antibodies Enhanced - Recombinant expressionⁱ Antibody validation by comparing the staining pattern of the antibody targeting the endogenous protein with that of a GFP-tagged recombinant version of the protein. Enhance validation is given when antibody staining of the endogenous protein overlaps with the tagged protein, potentially also with some additional localizations. Antibody staining overlaps with GFP tagged protein Immunofluorescent staining of transgenic HeLa cells show antibody staining in nucleoplasm & nuclear bodies and GFP expression in nucleoplasm & nuclear bodies.	N/A	N/A

Antibody dilution		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 GFP assay: Recombinant HeLa (BAC 4662) tested with dilution: 1:24	Human assay: HEK293 fixed with PFA, dilution: 1:125 Human assay: SH-SY5Y fixed with PFA, dilution: 1:125 Human assay: U2OS fixed with PFA, dilution: 1:125 GFP assay: Recombinant HeLa (BAC 4662) tested with dilution: 1:125
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.	Supportedⁱ 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 tonsil shows strong nuclear positivity in germinal center and non-germinal center cells. Tonsil	N/A	N/A	Enhanced - Orthogonal Antibody staining mainly consistent with RNA expression data across 43 tissues. HIGH EXPRESSION Epididymis RNA expression: 18.4 nTPM LOW EXPRESSION Cerebral cortex RNA expression: 19.2 nTPM	Enhanced - Orthogonal Antibody staining mainly consistent with RNA expression data across 44 tissues. HIGH EXPRESSION Epididymis RNA expression: 18.4 nTPM LOW EXPRESSION Cerebral cortex RNA expression: 19.2 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 pH6	HIER pH6
Antibody dilution	1:75			1:100	1:75
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.	Medium consistency between antibody staining and RNA expression data.			Medium consistency between antibody staining and RNA expression data.	Medium 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 - Capture MSⁱ This method is based on comparison between the molecular weight of the stained band visualized by the antibody against the protein size obtained by a capture MS method in which multiple gel slices are cut out from the electrophoretic separation of cell lysates of RT4 and U-251 and analysed separately by proteomics. The band detected by the antibody should be equivalent to the same of the intended target protein and its peptide(s). Antibody band pattern is confirmed by capture-MS. 230 130 95 72 56 36 28 17 11 Enhanced - Recombinant expressionⁱ This method is based on over-expression of the target protein in a cell line preferably not expressing the target protein. The staining of the antibody is evaluated by Western blot through analyses of samples from cell lysates with and without recombinant expression of the target protein. The results show no or weak band from the unmodified cell line lysate and a strong band in the cell line with recombinant expression. Single band corresponding to the predicted size in kDa (+/-20%). Lane 1: Marker [kDa] 250, 130, 95, 72, 55, 36, 28, 17, 10 Lane 2: Negative control (vector only transfected HEK293T lysate) Lane 3: Over-expression Lysate (Co-expressed with a C-terminal myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells, LY427987) Target mass (kDa): 82.9, 81.4, 76.3, 73.6	Enhanced - Independent antibodiesⁱ This method is based on comparing the staining pattern using two independent antibodies with no overlapping epitopes. The staining of the two antibodies is compared by Western blot through analyses of samples from at least two cell lysates preferably expressing the target protein at different levels. The results show similar Western Blot patterns achieved with independent antibodies. Antibody band pattern is confirmed by antibody HPA065779. Analysis performed using a standard panel of samples. 230 130 95 72 56 36 28 17 11	Enhanced - Independent antibodiesⁱ This method is based on comparing the staining pattern using two independent antibodies with no overlapping epitopes. The staining of the two antibodies is compared by Western blot through analyses of samples from at least two cell lysates preferably expressing the target protein at different levels. The results show similar Western Blot patterns achieved with independent antibodies. Antibody band pattern is confirmed by antibody HPA008797. Analysis performed using a standard panel of samples. 250 130 95 72 55 36 28 17 10	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. Weak band of predicted size but with additional bands of higher intensity also present. 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. Weak band of predicted size but with additional bands of higher intensity also present. Analysis performed using a standard panel of samples.

Antibody dilution	Capture MS: 1:250 Recombinant: 1:125	1:250	1:420	1:500	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.	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.	N/A	N/A

Antibody dilution	1:500	1:500	1:5000
RELEVANT PUBLICATIONS
	Altered telomeres in tumors with ATRX and DAXX mutations Heaphy CM et al Science 2011;333(6041):425
	Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma Schwartzentruber J et al Nature 2012;482(7384):226-31
	Loss of ATRX or DAXX expression and concomitant acquisition of the alternative lengthening of telomeres phenotype are late events in a small subset of MEN-1 syndrome pancreatic neuroendocrine tumors de Wilde RF et al Mod Pathol 2012;25(7):1033-9
	Sp100 provides intrinsic immunity against human papillomavirus infection Stepp WH et al mBio 2013;4(6):e00845-13
	Whole-exome sequencing identifies somatic ATRX mutations in pheochromocytomas and paragangliomas Fishbein L et al Nat Commun 2015;6:6140 Application: IHC
	Epigenetic dysregulation and poorer prognosis in DAXX-deficient pancreatic neuroendocrine tumours Pipinikas CP et al Endocr Relat Cancer 2015;22(3):L13-8 Application: IHC
	ATRX represses alternative lengthening of telomeres Napier CE et al Oncotarget 2015;6(18):16543-58 Application: WB
	Exome Sequencing of Uterine Leiomyosarcomas Identifies Frequent Mutations in TP53, ATRX, and MED12 Mäkinen N et al PLoS Genet 2016;12(2):e1005850 Application: IHC
	TERT promoter mutations in pancreatic endocrine tumours are rare and mainly found in tumours from patients with hereditary syndromes Vinagre J et al Sci Rep 2016;6:29714 Application: IHC
	Loss of ATRX and DAXX expression identifies poor prognosis for smooth muscle tumours of uncertain malignant potential and early stage uterine leiomyosarcoma Slatter TL et al J Pathol Clin Res 2015;1(2):95-105 Application: IHC
	Alternative Lengthening of Telomeres in Primary Pancreatic Neuroendocrine Tumors Is Associated with Aggressive Clinical Behavior and Poor Survival Kim JY et al Clin Cancer Res 2017;23(6):1598-1606 Application: IHC
	Daxx inhibits hypoxia-induced lung cancer cell metastasis by suppressing the HIF-1α/HDAC1/Slug axis Lin CW et al Nat Commun 2016;7:13867 Application: IHC
	Alternative lengthening of telomeres and ATRX/DAXX loss can be reliably detected in FNAs of pancreatic neuroendocrine tumors VandenBussche CJ et al Cancer Cytopathol 2017;125(7):544-551 Application: IHC
	Anti-proliferative and anti-secretory effects of everolimus on human pancreatic neuroendocrine tumors primary cultures: is there any benefit from combination with somatostatin analogs? Mohamed A et al Oncotarget 2017;8(25):41044-41063 Application: IHC
	DAXX/ATRX and MEN1 genes are strong prognostic markers in pancreatic neuroendocrine tumors Park JK et al Oncotarget 2017;8(30):49796-49806 Application: IHC
	ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner Brosnan-Cashman JA et al PLoS One 2018;13(9):e0204159 Application: IHC, WB
	Importance of Promyelocytic Leukema Protein (PML) for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication Hossain MG et al Front Microbiol 2018;9:2324 Application: ICC-IF, WB
	TERT promoter mutations are frequent and show association with MED12 mutations in phyllodes tumors of the breast Yoshida M et al Br J Cancer 2015;113(8):1244-8 Application: IHC
	Grading of Neuroendocrine Carcinomas: Correlation of Ga-PET/CT Scan with Tissue Biomarkers Liverani C et al Dis Markers 2018;2018:6878409 Application: IHC
	Alternative Lengthening of Telomeres and Loss of DAXX/ATRX Expression Predicts Metastatic Disease and Poor Survival in Patients with Pancreatic Neuroendocrine Tumors Singhi AD et al Clin Cancer Res 2017;23(2):600-609 Application: IHC
	A Comparison of Death Domain-Associated Protein 6 in Different Endometrial Carcinomas Histotypes Jin C et al Biomark Insights 2019;14:1177271919864892 Application: IHC
	Alternative lengthening of telomeres, ATRX loss and H3-K27M mutations in histologically defined pilocytic astrocytoma with anaplasia Rodriguez FJ et al Brain Pathol 2019;29(1):126-140 Application: IHC
ANTIGEN INFORMATION
Antigen	Recombinant protein fragment	Recombinant protein fragment	Recombinant protein fragment	Not known	Recombinant protein
Length (aa)	146	146	136
Antigen sequence	`DEEEEAAAGKDGDKSPMSSLQISNEKNLEPGKQISRSSGEQQNKGRIVSP SLLSEEPLAPSSIDAESNGEQPEELTLEEESPVSQLFELEIEALPLDTPS SVETDISSSRKQSEEPFTTVLENGAGMVSSTSFNGGVSPHNWGDSG`	`DEEEEAAAGKDGDKSPMSSLQISNEKNLEPGKQISRSSGEQQNKGRIVSP SLLSEEPLAPSSIDAESNGEQPEELTLEEESPVSQLFELEIEALPLDTPS SVETDISSSRKQSEEPFTTVLENGAGMVSSTSFNGGVSPHNWGDSG`	`ARGSSSSGGKKCYKLENEKLFEEFLELCKMQTADHPEVVPFLYNRQQRAH SLFLASAEFCNILSRVLSRARSRPAKLYVYINELCTVLKAHSAKKKLNLA PAATTSNEPSGNNPPTHLSLDPTNAENTASQSPRTR`
Matching transcripts	DAXX-201 - ENSP00000266000 [100%] DAXX-202 - ENSP00000363668 [100%] DAXX-203 - ENSP00000396876 [100%] DAXX-210 - ENSP00000482399 [100%] DAXX-211 - ENSP00000516212 [100%]	DAXX-201 - ENSP00000266000 [100%] DAXX-202 - ENSP00000363668 [100%] DAXX-203 - ENSP00000396876 [100%] DAXX-210 - ENSP00000482399 [100%] DAXX-211 - ENSP00000516212 [100%]	DAXX-201 - ENSP00000266000 [100%] DAXX-202 - ENSP00000363668 [100%] DAXX-210 - ENSP00000482399 [100%] DAXX-211 - ENSP00000516212 [100%] DAXX-204 - ENSP00000406008 [97%]
Matching mouse transcripts	ENSMUSP00000078390 [47%] ENSMUSP00000128504 [47%] ENSMUSP00000134158 [47%] ENSMUSP00000127870 [24%] ENSMUSP00000097965 [23%]	ENSMUSP00000078390 [47%] ENSMUSP00000128504 [47%] ENSMUSP00000134158 [47%] ENSMUSP00000127870 [24%] ENSMUSP00000097965 [23%]	ENSMUSP00000078390 [76%] ENSMUSP00000128504 [76%] ENSMUSP00000133552 [76%] ENSMUSP00000133303 [66%] ENSMUSP00000133319 [49%] ENSMUSP00000096096 [23%] ENSMUSP00000159204 [21%]
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.

DAXX-201 DAXX-202 DAXX-203 DAXX-204 DAXX-210 DAXX-211

Displaying protein features on the AlphaFold structures

The Protein Browser