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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-TFEB antibody [EPR22941-6] (ab267351)

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Western blot - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Western blot - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Flow Cytometry (Intracellular) - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)
  • Anti-TFEB antibody [EPR22941-6] (ab267351)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR22941-6] to TFEB
  • Suitable for: WB, IP, ICC/IF, IHC-P, Flow Cyt (Intra)
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-TFEB antibody [EPR22941-6]
    See all TFEB primary antibodies

  • Description

    Rabbit monoclonal [EPR22941-6] to TFEB

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IP, ICC/IF, IHC-P, Flow Cyt (Intra)more details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Raji and IM-9 lysates. IHC-P: Human colon, Human lung cancer and Human breast cancer tissues. ICC/IF: Raji and IM-9 cells. Flow Cyt (intra): IM-9 cells. IP: Raji and IM-9 cells.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    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

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab267351 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)
1/1000. Detects a band of approximately 66 kDa (predicted molecular weight: 53 kDa).

 

 
IP
1/30.
ICC/IF
1/50.

 

 
IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

 

 
Flow Cyt (Intra)
1/500.
Notes
WB
1/1000. Detects a band of approximately 66 kDa (predicted molecular weight: 53 kDa).

 

 
IP
1/30.
ICC/IF
1/50.

 

 
IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

 

 
Flow Cyt (Intra)
1/500.

Target

  • Function

    Transcription factor that specifically recognizes and binds E-box sequences (3'-CANNTG-5'). Efficient DNA-binding requires dimerization with itself or with another MiT/TFE family member such as TFE3 or MITF. In association with TFE3, activates the expression of CD40L in T-cells, thereby playing a role in T-cell-dependent antibody responses in activated CD4(+) T-cells and thymus-dependent humoral immunity. Specifically recognizes and binds the CLEAR-box sequence (5'-GTCACGTGAC-3') present in the regulatory region of many lysosomal genes, leading to activate their expression. It thereby plays a central role in expression of lysosomal genes. Specifically recognizes the gamma-E3 box, a subset of E-boxes, present in the heavy-chain immunoglobulin enhancer. Plays a role in the signal transduction processes required for normal vascularization of the placenta.

  • Sequence similarities

    Belongs to the MiT/TFE family.
    Contains 1 basic helix-loop-helix (bHLH) domain.

  • Domain

    The leucin zipper region is essential for homo- or heterodimerization and high-affinity DNA binding. DNA binding is mediated by the basic region.

  • Post-translational
    modifications

    Sumoylated; does not affect dimerization with MITF.

  • Cellular localization

    Cytoplasm. Nucleus. Mainly present in the cytoplasm. Under aberrant lysosomal storage conditions, it translocates from the cytoplasm to the nucleus.
  • Information by UniProt

  • Database links

    • Alternative names

      • Alpha TFEB antibody
      • AlphaTFEB antibody
      • bHLHe35 antibody
      • Class E basic helix-loop-helix protein 35 antibody
      • T cell transcription factor EB antibody
      • TCFEB antibody
      • TFEB antibody
      • TFEB_HUMAN antibody
      • Transcription factor EB antibody
      see all

    Images

    • Western blot - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Western blot - Anti-TFEB antibody [EPR22941-6] (ab267351)
      All lanes : Anti-TFEB antibody [EPR22941-6] (ab267351) at 1/1000 dilution

      Lane 1 : Wild-type HepG2 cell lysate
      Lane 2 : TFEB knockout HepG2 cell lysate
      Lane 3 : Raji cell lysate
      Lane 4 : MCF7 cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 53 kDa
      Observed band size: 60 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-TFEB antibody [EPR22941-6] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab267351 was shown to bind specifically to TFEB. A band was observed at 60 kDa in wild-type HepG2 cell lysates with no signal observed at this size in TFEB knockout cell line ab277871 (knockout cell lysate ab284220). To generate this image, wild-type and TFEB knockout HepG2 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

    • Western blot - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Western blot - Anti-TFEB antibody [EPR22941-6] (ab267351)
      All lanes : Anti-TFEB antibody [EPR22941-6] (ab267351) at 1/1000 dilution

      Lane 1 : Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate
      Lane 2 : IM-9 (human multiple myeloma B Lymphoblast whole cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

      Predicted band size: 53 kDa
      Observed band size: 66 kDa why is the actual band size different from the predicted?



      Blocking and dilution buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

      The molecular weight  observed is consistent with what has been described in the literature (PMID:30519051).

       

    • Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (ab267351)

      TFEB was immunoprecipitated from 0.35 mg IM-9 (human multiple myeloma B Lymphoblast) whole cell lysate with ab267351 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267351 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

      Lane 1: IM-9 (human multiple myeloma B Lymphoblast) whole cell lysate 10ug

      Lane 2: ab267351 IP in IM-9 whole cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267351 in IM-9 whole cell lysate

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 2 min.

      The weak band around 50kDa is one isoform of TFEB(PMID:28993453).

       

    • Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (ab267351)

      TFEB was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab267351 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267351 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

      Lane 1: Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate 10ug

      Lane 2: ab267351 IP in Raji whole cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267351 in Raji whole cell lysate

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 15 seconds.

      The weak band around 50kDa is one isoform of TFEB(PMID:28993453).

       

    • Flow Cytometry (Intracellular) - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Flow Cytometry (Intracellular) - Anti-TFEB antibody [EPR22941-6] (ab267351)

      Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized IM-9 (human multiple myeloma B Lymphoblast) cells labelling TFEB with ab267351 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. 

       

       

    • Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22941-6] (ab267351)

      Immunofluorescent analysis of 100% Methanol-fixed IM-9 (human multiple myeloma B Lymphoblast) cells labelling TFEB with ab267351 at 1/50 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in IM-9 cells. 100% methanol fixation is recommended. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

    • Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22941-6] (ab267351)

      Immunofluorescent analysis of 100% Methanol-fixed Raji (human Burkitt's lymphoma B lymphocyte) cells labelling TFEB with ab267351 at 1/50 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in Raji cells. 100% methanol fixation is recommended. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is  ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)

      Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling TFEB with ab267351 at 1/500 dilution (1.042ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human breast cancer (PMID:28017540). The section was incubated with ab256492 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND®RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)

      Immunohistochemical analysis of paraffin-embedded Human lung cancer tissue labeling TFEB with ab267351 at 1/500 dilution (1.042ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human lung cancer (PMID: 26264650) The section was incubated with ab256492 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (ab267351)

      Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling TFEB with ab267351 at 1/500 dilution (1.042ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining human colon (PMID:30519051). The section was incubated with ab256492 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

    • Anti-TFEB antibody [EPR22941-6] (ab267351)
      Anti-TFEB antibody [EPR22941-6] (ab267351)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References (0)

    Publishing research using ab267351? Please let us know so that we can cite the reference in this datasheet.

    ab267351 has not yet been referenced specifically in any publications.

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    Western blot abreview for Anti-TFEB antibody [EPR22941-6]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Pig Tissue lysate - whole (heart)
    Gel Running Conditions
    Non-reduced Denaturing (NU-PAGE 4-12 %)
    Loading amount
    50 µg
    Specification
    heart
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Weilan Mo

    Verified customer

    Submitted Aug 16 2021

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