Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-MUC1 antibody [EPR1023] - Low endotoxin, Azide free (ab194978)

Overview

  • Product name

    Anti-MUC1 antibody [EPR1023] - Low endotoxin, Azide free
    See all MUC1 primary antibodies
  • Description

    Rabbit monoclonal [EPR1023] to MUC1 - Low endotoxin, Azide free
  • Host species

    Rabbit
  • Specificity

    ab194978 detects the full-length MUC1 and the 17 kDa carboxy-terminal subunit (subunit beta).
  • Tested applications

    Suitable for: IHC-P, WB, IP, Flow Cyt, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human MUC1 aa 1200-1300 (C terminal). The exact sequence is proprietary.

  • Positive control

    • T47-D cell lysate, colon cancer lysate, Human breast carcinoma tissue, Human normal breast tissue.
  • General notes

    ab194978 is a carrier-free antibody designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our Low endotoxin, azide-free formats have low endotoxin level (≤ 1 EU/ml, determined by the LAL assay) and are free from azide, to achieve consistent experimental results in functional assays.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

Properties

Applications

Our Abpromise guarantee covers the use of ab194978 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use at an assay dependent concentration. Predicted molecular weight: 122 kDa.
IP Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF Use at an assay dependent concentration.

Target

  • Function

    The alpha subunit has cell adhesive properties. Can act both as an adhesion and an anti-adhesion protein. May provide a protective layer on epithelial cells against bacterial and enzyme attack.
    The beta subunit contains a C-terminal domain which is involved in cell signaling, through phosphorylations and protein-protein interactions. Modulates signaling in ERK, SRC and NF-kappa-B pathways. In activated T-cells, influences directly or indirectly the Ras/MAPK pathway. Promotes tumor progression. Regulates TP53-mediated transcription and determines cell fate in the genotoxic stress response. Binds, together with KLF4, the PE21 promoter element of TP53 and represses TP53 activity.
  • Tissue specificity

    Expressed on the apical surface of epithelial cells, especially of airway passages, breast and uterus. Also expressed in activated and unactivated T-cells. Overexpressed in epithelial tumors, such as breast or ovarian cancer and also in non-epithelial tumor cells. Isoform Y is expressed in tumor cells only.
  • Involvement in disease

    MUC1/CA 15-3 is used as a serological clinical marker of breast cancer to monitor response to breast cancer treatment and disease recurrence (PubMed:20816948). Decreased levels over time may be indicative of a positive response to treatment. Conversely, increased levels may indicate disease progression. At an early stage disease, only 21% of patients exhibit high MUC1/CA 15-3 levels, that is why CA 15-3 is not a useful screening test. Most antibodies target the highly immunodominant core peptide domain of 20 amino acid (APDTRPAPGSTAPPAHGVTS) tandem repeats. Some antibodies recognize glycosylated epitopes.
    Medullary cystic kidney disease 1
  • Sequence similarities

    Contains 1 SEA domain.
  • Developmental stage

    During fetal development, expressed at low levels in the colonic epithelium from 13 weeks of gestation.
  • Post-translational
    modifications

    Highly glycosylated (N- and O-linked carbohydrates and sialic acid). O-glycosylated to a varying degree on serine and threonine residues within each tandem repeat, ranging from mono- to penta-glycosylation. The average density ranges from about 50% in human milk to over 90% in T47D breast cancer cells. Further sialylation occurs during recycling. Membrane-shed glycoproteins from kidney and breast cancer cells have preferentially sialyated core 1 structures, while secreted forms from the same tissues display mainly core 2 structures. The O-glycosylated content is overlapping in both these tissues with terminal fucose and galactose, 2- and 3-linked galactose, 3- and 3,6-linked GalNAc-ol and 4-linked GlcNAc predominating. Differentially O-glycosylated in breast carcinomas with 3,4-linked GlcNAc. N-glycosylation consists of high-mannose, acidic complex-type and hybrid glycans in the secreted form MUC1/SEC, and neutral complex-type in the transmembrane form, MUC1/TM.
    Proteolytic cleavage in the SEA domain occurs in the endoplasmic reticulum by an autoproteolytic mechanism and requires the full-length SEA domain as well as requiring a Ser, Thr or Cys residue at the P + 1 site. Cleavage at this site also occurs on isoform MUC1/X but not on isoform MUC1/Y. Ectodomain shedding is mediated by ADAM17.
    Dual palmitoylation on cysteine residues in the CQC motif is required for recycling from endosomes back to the plasma membrane.
    Phosphorylated on tyrosines and serine residues in the C-terminal. Phosphorylation on tyrosines in the C-terminal increases the nuclear location of MUC1 and beta-catenin. Phosphorylation by PKC delta induces binding of MUC1 to beta-catenin/CTNNB1 and thus decreases the formation of the beta-catenin/E-cadherin complex. Src-mediated phosphorylation inhibits interaction with GSK3B. Src- and EGFR-mediated phosphorylation on Tyr-1229 increases binding to beta-catenin/CTNNB1. GSK3B-mediated phosphorylation on Ser-1227 decreases this interaction but restores the formation of the beta-cadherin/E-cadherin complex. On T-cell receptor activation, phosphorylated by LCK. PDGFR-mediated phosphorylation increases nuclear colocalization of MUC1CT and CTNNB1.
    The N-terminal sequence has been shown to begin at position 24 or 28.
  • Cellular localization

    Secreted; Cell membrane. Cytoplasm. Nucleus. On EGF and PDGFRB stimulation, transported to the nucleus through interaction with CTNNB1, a process which is stimulated by phosphorylation. On HRG stimulation, colocalizes with JUP/gamma-catenin at the nucleus and Apical cell membrane. Exclusively located in the apical domain of the plasma membrane of highly polarized epithelial cells. After endocytosis, internalized and recycled to the cell membrane. Located to microvilli and to the tips of long filopodial protusions.
  • Information by UniProt
  • Database links

  • Alternative names

    • ADMCKD antibody
    • ADMCKD1 antibody
    • Breast carcinoma associated antigen DF3 antibody
    • Breast carcinoma-associated antigen DF3 antibody
    • CA 15-3 antibody
    • CA15 3 antibody
    • CA15 3 antigen antibody
    • CA15-3 antibody
    • CA15.3 antibody
    • Cancer antigen 15-3 antibody
    • Carcinoma associated mucin antibody
    • Carcinoma-associated mucin antibody
    • CD 227 antibody
    • CD227 antibody
    • DF3 antigen antibody
    • EMA antibody
    • Episialin antibody
    • Epithelial Membrane Antigen antibody
    • H23 antigen antibody
    • H23AG antibody
    • KL 6 antibody
    • KL-6 antibody
    • KL6 antibody
    • Krebs von den Lungen-6 antibody
    • MAM 6 antibody
    • MAM6 antibody
    • MCD antibody
    • MCKD antibody
    • MCKD1 antibody
    • Medullary cystic kidney disease 1 (autosomal dominant) antibody
    • Medullary cystic kidney disease, autosomal dominant antibody
    • MUC 1 antibody
    • MUC-1 antibody
    • MUC-1/SEC antibody
    • MUC-1/X antibody
    • MUC1 antibody
    • MUC1-alpha antibody
    • MUC1-beta antibody
    • MUC1-CT antibody
    • MUC1-NT antibody
    • MUC1/ZD antibody
    • MUC1_HUMAN antibody
    • Mucin 1 antibody
    • Mucin 1 cell surface associated antibody
    • Mucin 1 transmembrane antibody
    • Mucin 1, cell surface associated antibody
    • Mucin-1 subunit beta antibody
    • Peanut reactive urinary mucin antibody
    • Peanut-reactive urinary mucin antibody
    • PEM antibody
    • PEMT antibody
    • Polymorphic epithelial mucin antibody
    • PUM antibody
    • Tumor associated epithelial membrane antigen antibody
    • Tumor associated epithelial mucin antibody
    • Tumor associated mucin antibody
    • Tumor-associated epithelial membrane antigen antibody
    • Tumor-associated mucin antibody
    see all

Images

  • Immunohistochemical staining of paraffin embedded rat kidney with purified ab109185 at a working dilution of 1 in 500. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • ab109185 (purified) at 1/20 immunoprecipitating MUC1 in T47-D (Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • Overlay histogram showing MCF7 cells fixed in 2% PFA and stained with purified ab109185 at a dilution of 1 in 30 (pink line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 150. Rabbit monoclonal IgG was used as an isotype control.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • Immunofluorescence staining of A431 cells with purified ab109185 at a working dilution of 1 in 1000, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 555 goat anti rabbit, used at a dilution of 1 in 400. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab109185 was used at a dilution of 1/200 followed by an Alexa Fluor® 555 goat anti-mouse antibody at a dilution of 1/500.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • Immunohistochemical staining of paraffin embedded mouse lung with purified ab109185 at a working dilution of 1 in 500. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • Immunohistochemical staining of paraffin embedded human endometrium with purified ab109185 at a working dilution of 1 in 500. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • Overlay histogram showing MCF7 cells stained with unpurified ab109185 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109185, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in MCF7 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • ab109185 (unpurified) showing positive staining in Normal stomach tissue.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • Equilibrium disassociation constant (KD) of unpurified ab109185.
    Learn more about KD

    Click here to learn more about KD

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109185).

  • All lanes : Anti-MUC1 antibody [EPR1023] (ab109185) at 1/1000 dilution

    Lane 1 : Wild-type HeLa whole cell lysate
    Lane 2 : MUC1 knockout HeLa whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 122 kDa



    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab194978).

    Lanes 1 - 2: Merged signal (red and green). Green - ab109185 observed at 24 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab109185 was shown to specifically react with MUC1 in wild-type HeLa cells as signal was lost in MUC1 knockout cells. Wild-type and MUC1 knockout samples were subjected to SDS-PAGE. Ab109185 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • This IHC data was generated using the same anti-MUC1 antibody clone [EPR1023] in a different buffer formulation (cat# ab109185).

    ab109185 (unpurified) showing positive staining in Breast ductal infiltrating carcinoma tissue.

References

This product has been referenced in:

  • Cyr AR  et al. TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis. Oncogene N/A:N/A (2014). Human . Read more (PubMed: 24469049) »
  • Wang WY  et al. A Gene Expression Signature of Epithelial Tubulogenesis and a Role for ASPM in Pancreatic Tumor Progression. Gastroenterology 145:1110-20 (2013). WB ; Human . Read more (PubMed: 23896173) »
See all 2 Publications for this product

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