Recombinant 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
Related conjugates and formulations
Overview
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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.
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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.
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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
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
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR22941-6 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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 |
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WB | (1) |
1/1000. Detects a band of approximately 66 kDa (predicted molecular weight: 53 kDa).
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IP |
1/30.
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ICC/IF |
1/50.
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IHC-P |
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Flow Cyt (Intra) |
1/500.
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Notes |
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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
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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
modificationsSumoylated; 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
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Database links
- Entrez Gene: 7942 Human
- Omim: 600744 Human
- SwissProt: P19484 Human
- Unigene: 485360 Human
- Unigene: 715310 Human
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Alternative names
- Alpha TFEB antibody
- AlphaTFEB antibody
- bHLHe35 antibody
see all
Images
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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.
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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).
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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).
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (2)
ab267351 has been referenced in 2 publications.
- An JH et al. Ceria-Zirconia nanoparticles reduce intracellular globotriaosylceramide accumulation and attenuate kidney injury by enhancing the autophagy flux in cellular and animal models of Fabry disease. J Nanobiotechnology 20:125 (2022). PubMed: 35264192
- Hu P et al. Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218. Acta Pharm Sin B 11:3178-3192 (2021). PubMed: 34729308