Overview

  • Product name
    Anti-Melanoma gp100 antibody [EP4863(2)]
    See all Melanoma gp100 primary antibodies
  • Description
    Rabbit monoclonal [EP4863(2)] to Melanoma gp100
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human Melanoma gp100 aa 1-100. The exact sequence is proprietary.
    Database link: P40967

  • Positive control
    • IHC-P: Human melanoma tissue. ICC/IF: B16-F0 mouse skin cells. WB: Human melanoma lysate; B16-F0 lysate. Flow Cyt: B16-F0 cells; MALME 3M cells. IP: B16-F0 whole cell lysate,
  • General notes

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab137078 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
WB 1/1000 - 1/10000. Predicted molecular weight: 70 kDa.
IHC-P 1/100 - 1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/250 - 1/500.
IP 1/10 - 1/20.
Flow Cyt 1/80.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Plays a central role in the biogenesis of melanosomes. Involved in the maturation of melanosomes from stage I to II. The transition from stage I melanosomes to stage II melanosomes involves an elongation of the vesicle, and the appearance within of distinct fibrillar structures. Release of the soluble form, ME20-S, could protect tumor cells from antibody mediated immunity.
  • Tissue specificity
    Preferentially expressed in melanomas. Some expression was found in dysplastic nevi. Not found in normal tissues nor in carcinomas. Normally expressed at low levels in quiescent adult melanocytes but overexpressed by proliferating neonatal melanocytes and during tumor growth.
  • Sequence similarities
    Belongs to the PMEL/NMB family.
    Contains 1 PKD domain.
  • Domain
    The RPT domain is essential for the generation of the fibrillar matrix of melanosomes.
    The lumenal domain is necessary for correct processing and trafficking to melanosomes.
  • Post-translational
    modifications
    A small amount of P1/P100 (major form) undergoes glycosylation to yield P2/P120 (minor form). P2 is cleaved by a furin-like proprotein convertase (PC) in a pH-dependent manner in a post-Golgi, prelysosomal compartment into two disulfide-linked subunits: a large lumenal subunit, M-alpha/ME20-S, and an integral membrane subunit, M-beta. Despite cleavage, only a small fraction of M-alpha is secreted, whereas most M-alpha and M-beta remain associated with each other intracellularly. M-alpha is further processed to M-alpha N and M-alpha C. M-alpha C further undergoes processing to yield M-alpha C1 and M-alpha C3 (M-alpha C2 in the case of PMEL17-is or PMEL17-ls). Formation of intralumenal fibrils in the melanosomes requires the formation of M-alpha that becomes incorporated into the fibrils. Stage II melanosomes harbor only Golgi-modified Pmel17 fragments that are derived from M-alpha and that bear sialylated O-linked oligosaccharides.
    N-glycosylated. O-glycosylated; contains sialic acid.
  • Cellular localization
    Secreted and Endoplasmic reticulum membrane. Golgi apparatus. Melanosome. Endosome > multivesicular body. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. Localizes predominantly to intralumenal vesicles (ILVs) within multivesicular bodies. Associates with ILVs found within the lumen of premelanosomes and melanosomes and particularly in compartments that serve as precursors to the striated stage II premelanosomes.
  • Information by UniProt
  • Database links
  • Alternative names
    • 95 kDa melanocyte specific secreted glycoprotein antibody
    • 95 kDa melanocyte-specific secreted glycoprotein antibody
    • D12S53E antibody
    • gp100 antibody
    • M-beta antibody
    • ME20 antibody
    • ME20 M/ME20 S antibody
    • ME20-M antibody
    • ME20-S antibody
    • ME20M antibody
    • ME20M/ME20S antibody
    • ME20S antibody
    • Melanocyte lineage specific antigen GP100 antibody
    • Melanocyte protein mel 17 antibody
    • Melanocyte protein Pmel 17 antibody
    • Melanocyte protein Pmel 17 precursor antibody
    • Melanocytes lineage-specific antigen GP100 antibody
    • Melanoma associated ME20 antigen antibody
    • Melanoma gp100 antibody
    • Melanoma-associated ME20 antigen antibody
    • Melanosomal matrix protein 17 antibody
    • Melanosomal matrix protein17 antibody
    • P1 antibody
    • p100 antibody
    • p26 antibody
    • PMEL 17 antibody
    • PMEL antibody
    • PMEL_HUMAN antibody
    • PMEL17 antibody
    • Premelanosome protein antibody
    • Secreted melanoma-associated ME20 antigen antibody
    • SI antibody
    • SIL antibody
    • SILV antibody
    • Silver (mouse homolog) like antibody
    • Silver homolog antibody
    • Silver locus protein homolog antibody
    • Silver, mouse, homolog of antibody
    see all

Images

  • All lanes : Anti-Melanoma gp100 antibody [EP4863(2)] (ab137078) at 1/1000 dilution

    Lane 1 : Human melanoma
    Lane 2 : B16-F0 (mouse skin) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 70 kDa



    Blocking buffer: 5% NFDM /TBST 

    Diluting buffer: 5% NFDM /TBST

  • ab137078 staining Melanoma gp100 in human melanoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

    Negative control 1: PBS in place of primary antibody.

  • ab137078 staining Melanoma gp100 in B16-F0 (mouse skin) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab7291 and ab150120 were used as counterstains for primary antibody ab137078 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

    Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
    Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

  • ab137078 staining Melanoma gp100 in the mouse cell line B16-F0 (mouse skin) by flow cytometry. Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% PBS-Tween for 20 min. The sample was incubated with the primary antibody at a dilution of 1/80. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/500 was used as the secondary antibody.

    Isoytype control: Rabbit monoclonal IgG (Black)

    Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

  • ab137078 immunoprecipitating Melanoma gp100. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/20 and VeriBlot for IP secondary antibody (HRP) (ab131366) at a dilution of 1/10000.

    Lane 1: B16-F0 (mouse skin) whole cell lysate 10ug
    Lane 2: B16-F0 (mouse skin) whole cell lysate
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab137078 in B16-F0 whole cell lysate

  • All lanes : Anti-Melanoma gp100 antibody [EP4863(2)] (ab137078) at 1/1000 dilution

    Lane 1 : B16-F0 lysate
    Lane 2 : Human melanoma lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : goat anti-rabbit HRP at 1/2000 dilution

    Predicted band size: 70 kDa

  • Overlay histogram showing MALME 3M cells stained with ab137078 (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 (ab137078, 1/100 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) (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.
  • Overlay histogram showing B16-F0 (Mouse skin melanoma cell) cells stained with ab137078 (red line). The cells were fixed with 2% paraformaldehyde and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated with ab137078 at 1/450 dilution. The secondary antibody used was Goat anti rabbit IgG (FITC) at 1/150 dilution. Isotype control antibody (black line) was Rabbit monoclonal IgG (ab172730). Unlabelled control (blue line) was cells without incubation with primary antibody and secondary antibody. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

References

This product has been referenced in:
  • Rosato PC  et al. Virus-specific memory T cells populate tumors and can be repurposed for tumor immunotherapy. Nat Commun 10:567 (2019). Read more (PubMed: 30718505) »
  • Commerford CD  et al. Mechanisms of Tumor-Induced Lymphovascular Niche Formation in Draining Lymph Nodes. Cell Rep 25:3554-3563.e4 (2018). Read more (PubMed: 30590031) »
See all 15 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Subcutaneous melanoma primary tumor B16F10)
Permeabilization
Yes - 1% Triton X-100 in TBS for 30min @ RT
Specification
Subcutaneous melanoma primary tumor B16F10
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 24°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Dec 12 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Dog Tissue sections (skin)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH9
Permeabilization
Yes - Tween-20
Specification
skin
Blocking step
RTU Power Vision blocking from Leica as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jun 04 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (skin melanoma)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: pH6 citrate buffer
Permeabilization
Yes - Tween-20
Specification
skin melanoma
Blocking step
BSA as blocking agent for 40 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Oct 12 2016

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Hair Follicle)
Permeabilization
No
Specification
Hair Follicle
Blocking step
Serum as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 10% · Temperature: 21°C
Fixative
Acetone

Abcam user community

Verified customer

Submitted Sep 06 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Skin melanoma)
Antigen retrieval step
Heat mediated
Permeabilization
No
Specification
Skin melanoma
Blocking step
Endogenous Peroxidase Block as blocking agent for 5 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Nov 02 2015

Application
Immunohistochemistry (Frozen sections)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 20°C
Sample
Mouse Tissue sections (skin tumor (Melanoma))
Specification
skin tumor (Melanoma)
Permeabilization
Yes - PBS with Triton-X 100
Fixative
Paraformaldehyde

Otataren

Verified customer

Submitted Feb 25 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Dako Flex peroxidase as blocking agent for 5 minute(s) · Concentration: 100% · Temperature: RT°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: High pH
Sample
Human Tissue sections (Malignant Melanoma)
Specification
Malignant Melanoma
Permeabilization
No
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jan 06 2015

Answer

Thank you for contacting us.

Regarding your second question about anti-Pmel17 ab117853, clone 7E3, I am waiting for the lab to send a western blot protocol. I expect it will be standard. Can you tell me how much sample you are loading per lane of the gel, how you block the membrane, and how much antibody you incubate with the blot?

Regarding ab137078, this is raised against a peptide located within the first 100 amino acids of the N terminus, so it should be capable of detecting your protein.

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