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ANTIBODY INFORMATION VALIDATION SUMMARY IMMUNOCYTOCHEMISTRY IMMUNOHISTOCHEMISTRY WESTERN BLOT PROTEIN ARRAY ANTIGEN INFORMATION RELEVANT PUBLICATIONS

ANTIBODY INFORMATION

 
 

Antibody HPA004712

 

Antibody CAB019417

 

Provider

Atlas Antibodies
Sigma-Aldrich
  Cell Signaling Technology, Inc
 

Product name

HPA004712   3113  

Host species

Rabbit   Rabbit  

Clonalityi

The antibodies are designated mAB for monoclonal and pAb for polyclonal.

pAb   mAb  

Purity

Affinity purified using the PrEST-antigen as affinity ligand   Not known  

Other gene matchi

Genes, excluding the target gene, to which the antigen sequence of the antibody is >80% identical.

   

Released in versioni

The release of the Human Protein Atlas in which the antibody was first published.

3   5  

Referencesi

References to publications in which the antibody has been used.

2    

VALIDATION SUMMARYi

Read more

 
 

Antibody HPA004712

 

Antibody CAB019417

 
Immunocytochemistryi
Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.

Read more

Supported    
Immunohistochemistryi
Immunohistochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in normal human tissues. For validation of antibody staining the immunohistochemical staining patterns obtained using tissue microarrays (TMA) containing 44 different normal tissue types (IHC tissue) , as well as, cell microarrays (CMA) containing 44 different widely used and well characterized human cell lines (IHC cells) , are compared to staining patterns from independent antibodies or to RNA expression patterns. For validation of protein expression patterns in normal human tissues the TMA staining patterns are evaluated together with RNA-seq data from internal and external sources and available protein/gene characterization data.

Read more

Supported  

Validated
 
Western bloti
A Western blot analysis is performed on a panel of human tissues and cell lines to evaluate antibody specificity. For antibodies with uncertain result a revalidation using an over-expression lysate is performed.

Read more

Supported   Uncertain  
Protein arrayi
A protein microarray containing 384 different antigens including the antibody target is used for analysis of antibody specificity.

Read more

Supported    

IMMUNOCYTOCHEMISTRYi

Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.

Read more

 
 

Antibody HPA004712

 

Antibody CAB019417

 

Standard validationi

For each antibody, the observed staining in the different cell lines is assigned a validation score based on concordance with available experimental gene/protein characterization data in the UniProtKB/Swiss-Prot database. The validation scores for up to three cell lines are merged into one of the main categories; Supported, Approved, or Uncertain to represent the overall antibody staining in all analyzed cell lines.

Read more

Supported    

   

Figure description

Immunofluorescent staining of human cell line CACO-2 shows localization to nucleoplasm.    

Antibody dilution

1:90    

Literature conformityi

The immunostaining patterns is compared for consistency with available experimental gene/protein characterization data in the UniProtKB/Swiss-Prot database and other experimental evidence for location described in scientific literature.

Read more

The subcellular location is supported by literature.    

IMMUNOHISTOCHEMISTRYi

Immunohistochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in normal human tissues. For validation of antibody staining the immunohistochemical staining patterns obtained using tissue microarrays (TMA) containing 44 different normal tissue types (IHC tissue) , as well as, cell microarrays (CMA) containing 44 different widely used and well characterized human cell lines (IHC cells) , are compared to staining patterns from independent antibodies or to RNA expression patterns. For validation of protein expression patterns in normal human tissues the TMA staining patterns are evaluated together with RNA-seq data from internal and external sources and available protein/gene characterization data.

Read more

 
 

Antibody HPA004712

 

Antibody CAB019417

 

Formal validation: Orthogonali

Formal validation based on orthogonal methods using cell line data.

Go to Data from the Human Protein Atlas in XML format to download cell line protein expression data and images.

Read more

 
Validated

Pearson correlation >0.6 for protein and RNA expression in cell lines.
 

 
 

 

 

Figure description

  Distribution of protein expression (antibody staining) and RNA expression (TPM) across 44 cell lines.  
 

Formal validation: Orthogonali

Formal validation based on orthogonal methods using tissue data.

Read more

 
Validated

Spearman correlation >0.6 for protein and RNA expression in tissues.
 

 
 

 

 

Figure description

  Distribution of protein expression (antibody staining) and RNA expression (TPM) across 37 tissues.  
 

Standard validationi

For each antibody the observed staining pattern is assigned a validation score based on the conformance with UniProt gene/protein characterization data, as well as, the consistency with RNA expression. The validation score levels are supported, approved, and uncertain.

Read more

Supported   Supported  

   

Figure description

Immunohistochemical staining of human small intestine shows strong nuclear positivity in glandular cells.   Immunohistochemical staining of human rectum shows nuclear positivity in glandular cells.  

Expression

RNA: detected in 13 tissues
Protein: detected in 52 cell types
  RNA: detected in 13 tissues
Protein: detected in 17 cell types
 

Retrieval

HIER pH6   HIER pH6  

Antibody dilution

1:75   1:50  

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.  

RNA consistencyi

Consistency between immunohistochemistry data and internally generated RNA-seq data is divided into five different categoreies: i) Consistent with RNA expression data, ii) Mainly consistent with RNA expression data, iii) Mainly not consistent with RNA expression data, iv) Not consistent with RNA expression data, and v) No internal RNA expression data available for correlation.

Mainly consistent with RNA expression data.   Mainly consistent with 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.

Read more

 
 

Antibody HPA004712

 

Antibody CAB019417

 

Standard 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 Supported, Approved, or Uncertain.

Read more

Supported

Single band corresponding to the predicted size in kDa (+/-20%).
  Uncertain

Analysis performed using a standard panel of samples.
Only bands not corresponding to the predicted size.
 

   

Figure description

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, LY400161)
   

Target mass (kDa)

52.8, 51.6, 50.1, 50, 49   52.8, 51.6, 50.1, 50, 49, 46.6, 44, 11.5  

Antibody dilution

1:250   1:500  

PROTEIN ARRAYi

A protein microarray containing 384 different antigens including the antibody target is used for analysis of antibody specificity.

Read more

 
 

Antibody HPA004712

 

Antibody CAB019417

 

Standard 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.

Read more

Supported

Pass with single peak corresponding to interaction only with its own antigen.
   

   

Figure description

Antibody specificity analysis with protein arrays. Predicted and matching interactions are shown in green.    

Antibody dilution

1:3000    

ANTIGEN INFORMATION

 
 

Antibody HPA004712

 

Antibody CAB019417

 

Antigen

Recombinant protein fragment   Synthetic peptide  

Length (aa)

105    

Antigen sequence

LLQEMLLGGSPSDAPHAHHPLHPHLMQEHMGTNVIVANTMPTHLSNGQMC
EWPRPRGQAATPETPQPSPPGGSGSEPYKLLPGAVATIVKPLSAIPQPTI
TKQEV
 
 

Matching transcripts

HNF4A-001 - ENSP00000315180 [100%]
HNF4A-002 - ENSP00000312987 [100%]
HNF4A-201 - ENSP00000481331 [100%]
HNF4A-005 - ENSP00000396216 [90%]
HNF4A-006 - ENSP00000412111 [90%]
   

Other gene matchi

Genes, excluding the target gene, to which the antigen sequence of the antibody is >80% identical.

   
ANTIGEN VIEW
HNF4A-001
HNF4A-002
HNF4A-005
HNF4A-006
HNF4A-201

RELEVANT PUBLICATIONS

 
 

Antibody HPA004712

 

Antibody CAB019417

 

1.

Global gene expression responses to low- or high-dose radiation in a human three-dimensional tissue model
Mezentsev A et al
Radiat Res 2011;175(6):677-88
   

2.

Inhibition of the glucose transporter SGLT2 with dapagliflozin in pancreatic alpha cells triggers glucagon secretion
Bonner C et al
Nat Med 2015;21(5):512-7
Application: ICC-IF
   
 

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