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MiTF Recombinant Rabbit Monoclonal Antibody [JF100-01]

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Specification

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Catalog# ET1702-86

MiTF Recombinant Rabbit Monoclonal Antibody [JF100-01]

Application

  • WB

  • IF-Cell

  • FC

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

MiTF Recombinant Rabbit Monoclonal Antibody [JF100-01]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Recombinant protein within Human MiTF aa 353-520 / 526.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell, FC

Molecular Weight

Predicted band size: 59 kDa

Positive Control

SK-MEL-28 cell lysate, HeLa cell lysate, A375 cell lysate, A172 cell lysate, B16-F1 cell lysate, PC-12 cell lysate, Hela, A431, NIH/3T3, SW480.

Conjugation

unconjugated

Clone Number

JF100-01

RRID

AB_3070346

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000

  • IF-Cell

  • 1:50-1:100

  • FC

  • 1:50-1:100

Target

Function

MITF (microphthalmia-associated transcription factor) is a melanocytic nuclear protein that contains basic helix-loop-helix (HLH) and leucine zipper (LZ) domains. These protein motifs are frequently observed in other transcription factors and are particularly common to members of the Myc family. MITF can directly associate with DNA as a homodimer and is required for the development and differentiation of melanocytes. Its expression is upregulated by cAMP and cAMP-dependent pathways. MITF activates several different gene promoters by binding to their E-boxes. Tyrosinase, TRP1 and TRP2 are pigment synthesis genes activated by MITF. When MITF is phosphorylated on Ser73 (via the MAPK pathway), it associates with co-activators of the p300/CBP family and enhances transcription. MITF has several isoforms including MITF-M which is specifically expressed in melanocytes. In MITF-deficient mice there is a complete absence of melanocytes.

Background References

1. Stemig M et al. Deletion of histone deacetylase 7 in osteoclasts decreases bone mass in mice by interactions with MITF. PLoS One 10:e0123843 (2015).

2. Sohn EH et al. Allogenic iPSC-derived RPE cell transplants induce immune response in pigs: a pilot study. Sci Rep 5:11791 (2015).

Sequence Similarity

Belongs to the MiT/TFE family.

Tissue Specificity

Isoform M is exclusively expressed in melanocytes and melanoma cells. Isoform A and isoform H are widely expressed in many cell types including melanocytes and retinal pigment epithelium (RPE). Isoform C is expressed in many cell types including RPE but not in melanocyte-lineage cells. Isoform Mdel is widely expressed in melanocytes, melanoma cell lines and tissues, but almost undetectable in non-melanoma cell lines.

Post-translational Modification

Phosphorylation at Ser-405 significantly enhances the ability to bind the tyrosinase promoter. Phosphorylated at Ser-180 and Ser-516 following KIT signaling, trigerring a short live activation: Phosphorylation at Ser-180 and Ser-516 by MAPK and RPS6KA1, respectively, activate the transcription factor activity but also promote ubiquitination and subsequent degradation by the proteasome.; Ubiquitinated following phosphorylation at Ser-180, leading to subsequent degradation by the proteasome. Deubiquitinated by USP13, preventing its degradation.

Subcellular Location

Nucleus, Cytoplasm.

UNIPROT

SwissProt: O75030 Human

SwissProt: Q08874 Mouse

SwissProt: O88368 Rat

Synonyms

BHLHE32 antibody

bHLHe32 antibody

Class E basic helix-loop-helix protein 32 antibody

CMM8 antibody

Homolog of mouse microphthalmia antibody

Mi antibody

Microphthalmia associated transcription factor antibody

Microphthalmia, mouse, homolog of antibody

Microphthalmia-associated transcription factor antibody

MITF antibody

Expand

Images

  • Western blot analysis of MiTF on different lysates with Rabbit anti-MiTF antibody (<a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a>) at 1/1,000 dilution.<br /><br />Lane 1: SK-MEL-28 cell lysate (20 µg/Lane)<br />Lane 2: HeLa cell lysate (20 µg/Lane)<br />Lane 3: A375 cell lysate (20 µg/Lane)<br />Lane 4: A172 cell lysate (20 µg/Lane)<br />Lane 5: B16-F1 cell lysate (20 µg/Lane)<br />Lane 6: PC-12 cell lysate (20 µg/Lane)<br /><br />Predicted band size: 59 kDa<br />Observed band size: 50 kDa<br /><br />Exposure time: 5 minutes;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of MiTF on different lysates with Rabbit anti-MiTF antibody (ET1702-86) at 1/1,000 dilution.

    Lane 1: SK-MEL-28 cell lysate (20 µg/Lane)
    Lane 2: HeLa cell lysate (20 µg/Lane)
    Lane 3: A375 cell lysate (20 µg/Lane)
    Lane 4: A172 cell lysate (20 µg/Lane)
    Lane 5: B16-F1 cell lysate (20 µg/Lane)
    Lane 6: PC-12 cell lysate (20 µg/Lane)

    Predicted band size: 59 kDa
    Observed band size: 50 kDa

    Exposure time: 5 minutes;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1702-86) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Knockdown (KD)</span><br /><br />Western blot analysis of MiTF on different lysates with Rabbit anti-MiTF antibody (<a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a>) at 1/1,000 dilution.<br /><br />Lane 1: Hela-si NT cell lysate (10 µg/Lane)<br />Lane 2: Hela-si MiTF cell lysate (10 µg/Lane)<br /><br />Predicted band size: 59 kDa<br />Observed band size: 55 kDa<br /><br />Exposure time: 3 minutes 26 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br /><a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a> was shown to specifically react with MiTF in Hela-si NT cells. Weakened band was observed when Hela-si MiTF sample was tested. Hela-si NT and Hela-si MiTF samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a>, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH, <a href="/products/ET1601-4" style="font-weight: bold;text-decoration: underline;">ET1601-4</a>, 1/10,000) were used in 5% BSA at 4 ℃ overnight. Goat Anti-rabbit IgG-HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/100,000 dilution was used for 1 hour at room temperature.

    ☑ Knockdown (KD)

    Western blot analysis of MiTF on different lysates with Rabbit anti-MiTF antibody (ET1702-86) at 1/1,000 dilution.

    Lane 1: Hela-si NT cell lysate (10 µg/Lane)
    Lane 2: Hela-si MiTF cell lysate (10 µg/Lane)

    Predicted band size: 59 kDa
    Observed band size: 55 kDa

    Exposure time: 3 minutes 26 seconds;

    4-20% SDS-PAGE gel.

    ET1702-86 was shown to specifically react with MiTF in Hela-si NT cells. Weakened band was observed when Hela-si MiTF sample was tested. Hela-si NT and Hela-si MiTF samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1702-86, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) were used in 5% BSA at 4 ℃ overnight. Goat Anti-rabbit IgG-HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.

  • ICC staining of MiTF in Hela cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;594 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of MiTF in Hela cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1702-86, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®594 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • ICC staining of MiTF in A431 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;594 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of MiTF in A431 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1702-86, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®594 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • ICC staining of MiTF in NIH/3T3 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;594 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of MiTF in NIH/3T3 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1702-86, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®594 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • Flow cytometric analysis of MiTF was done on SW480 cells. The cells were fixed, permeabilized and stained with the primary antibody (<a href="/products/ET1702-86" style="font-weight: bold;text-decoration: underline;">ET1702-86</a>, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of MiTF was done on SW480 cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1702-86, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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Citation