BRD4 - Antibodies - The Human Protein Atlas


	Antibody HPA015055	Antibody HPA061646	Antibody CAB068177	Antibody CAB068178

ANTIBODY INFORMATION
Provider	Atlas Antibodies Sigma-Aldrich	Atlas Antibodies Sigma-Aldrich	Atlas Antibodies	Atlas Antibodies
Product name	HPA015055	HPA061646	AMAb90841	AMAb90843
Host species	Rabbit	Rabbit	Mouse	Mouse
Clonalityⁱ The antibodies are designated mAB for monoclonal and pAb for polyclonal.	pAb	pAb	mAb	mAb
Concentration	0.0525 mg/ml	0.2248 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	Protein A/G	Protein A/G
Released in versionⁱ The release of the Human Protein Atlas in which the antibody was first published.	4.1	13.0	13.0	13.0
Referencesⁱ References to publications in which the antibody has been used.	5	2
Proper citation	Atlas Antibodies Cat#HPA015055, RRID:AB_1845435	Atlas Antibodies Cat#HPA061646, RRID:AB_2684575	Atlas Antibodies Cat#AMAb90841, RRID:AB_2665685	Atlas Antibodies Cat#AMAb90843, RRID:AB_2665686
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 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.	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 HPA061646. Immunofluorescent staining of human cell line U-251MG shows localization to nucleoplasm. 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 HPA015055. Immunofluorescent staining of human cell line PC-3 shows localization to nucleoplasm. Protein location across all samples similar between independent antibodies	N/A	N/A

Antibody dilution	Human assay: U-251MG fixed with PFA, dilution: 1:26 Human assay: U2OS fixed with PFA, dilution: 1:26	Human assay: MCF-7 fixed with PFA, dilution: 1:112 Human assay: PC-3 fixed with PFA, dilution: 1:112 Human assay: U2OS fixed with PFA, dilution: 1:112
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 cerebellum shows strong cytoplasmic positivity in purkinje cells. Cerebellum	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 skin shows strong nuclear positivity in epidermal cells. Skin 2	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 colon shows moderate nuclear positivity in glandular cells. Colon	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 kidney shows strong nuclear positivity in cells in tubules and cells in glomeruli. Kidney

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	HIER pH6
Antibody dilution	1:35	1:250	1:250	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 extensive gene/protein characterization data.	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.	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.	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. Only bands not corresponding to the predicted size. Analysis performed using a standard panel of samples.	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. 250 130 95 72 55 36 28 17 10	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	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. No bands detected. Analysis performed using a standard panel of samples. 250 130 100 70 55 35 25 15 10

Antibody dilution	1:250	1:370	1:1000	1:1000
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.	N/A	N/A

Antibody dilution	1:3000	1:4500
RELEVANT PUBLICATIONS
	RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia Zuber J et al Nature 2011;478(7370):524-8	miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein Tonouchi E et al Sci Rep 2018;8(1):4482
	Suppression of BRD4 inhibits human hepatocellular carcinoma by repressing MYC and enhancing BIM expression Li GQ et al Oncotarget 2016;7(3):2462-74 Application: IHC	Tumoral BRD4 expression in lymph node-negative breast cancer: association with T-bet+ tumor-infiltrating lymphocytes and disease-free survival Lee M et al BMC Cancer 2018;18(1):750 Application: IHC
	Automated Learning of Subcellular Variation among Punctate Protein Patterns and a Generative Model of Their Relation to Microtubules Johnson GR et al PLoS Comput Biol 2015;11(12):e1004614 Application: ICC-IF
	Regulation of the glucocorticoid receptor via a BET-dependent enhancer drives antiandrogen resistance in prostate cancer Shah N et al Elife 2017;6: Application: ChIP
	A Model System for Studying the DNMT3A Hotspot Mutation (DNMT3A) Demonstrates a Causal Relationship between Its Dominant-Negative Effect and Leukemogenesis Lu R et al Cancer Res 2019;79(14):3583-3594 Application: ChIP
ANTIGEN INFORMATION
Antigen	Recombinant protein fragment	Recombinant protein fragment	Recombinant protein	Recombinant protein
Length (aa)	142	78
Antigen sequence	`PQPAKPQQVIQHHHSPRHHKSDPYSTGHLREAPSPLMIHSPQMSQFQSLT HQSPPQQNVQPKKQELRAASVVQPQPLVVVKEEKIHSPIIRSEPFSPSLR PEPPKHPESIKAPVHLPQRPEMKPVDVGRPVIRPPEQNAPPP`	`PPFIATQVPVLEPQLPGSVFDPIGHFTQPILHLPQPELPPHLPQPPEHST PPHLNQHAVVSPPALHNALPQQPSRPSN`
Matching transcripts	BRD4-201 - ENSP00000263377 [100%] BRD4-213 - ENSP00000506350 [100%]	BRD4-201 - ENSP00000263377 [100%] BRD4-213 - ENSP00000506350 [100%]
Matching mouse transcripts	ENSMUSP00000155477 [77%] ENSMUSP00000003726 [76%] ENSMUSP00000113070 [76%] ENSMUSP00000135942 [30%] ENSMUSP00000153765 [30%]	ENSMUSP00000003726 [97%] ENSMUSP00000113070 [97%] ENSMUSP00000097901 [36%] ENSMUSP00000098591 [35%]
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.

BRD4-201 BRD4-213

Displaying protein features on the AlphaFold structures

The Protein Browser