TFEB Antibody

What is TFEB and why is it significant in research?

TFEB (Transcription Factor EB) is a protein encoded by the TFEB gene (ID: 7942) with a calculated molecular weight of approximately 53 kDa, though it typically appears at 65-70 kDa in gel electrophoresis due to post-translational modifications . TFEB plays critical roles in cellular processes including lysosomal biogenesis, autophagy regulation, and immune cell function. Recent research has identified TFEB as a hallmark of antigenic experience in B lymphocytes, making it particularly significant for immunological studies .

What types of TFEB antibodies are available for research applications?

Researchers can access several types of TFEB antibodies, including monoclonal antibodies (mouse or rabbit host) that target specific epitopes, and polyclonal antibodies that recognize multiple epitopes. Available options include Mouse Anti-Human TFEB Monoclonal Antibody (Clone #954604) , Rabbit Monoclonal (E5P9M) , standard Rabbit Antibody , and polyclonal antibodies . These come in both unconjugated forms or paired with detection systems like HRP-conjugated or fluorescence-conjugated secondary antibodies for different visualization needs .

Which applications are suitable for TFEB antibody detection?

TFEB antibodies have been validated across multiple applications including:

• Western Blotting (WB) at dilutions typically ranging from 1:1000-1:6000

• Immunoprecipitation (IP) at approximately 1:200 dilution

• Chromatin Immunoprecipitation (ChIP) at 1:50 dilution

• Immunohistochemistry (IHC) at 1:50-1:500 dilution

• Immunofluorescence/Immunocytochemistry (IF/ICC) at 1:50-1:500 dilution

The specific dilution should be optimized for each experimental system to obtain optimal results .

How should I optimize Western blot protocols for detecting TFEB?

For optimal TFEB detection via Western blot:

• Sample preparation: Use appropriate lysis buffer for your cell/tissue type (examples from search results include Raji human Burkitt's lymphoma cell line, A549 human lung carcinoma, and various tissues like brain and heart)

• Protein loading: Load sufficient protein (typically 10-30 μg of total protein)

• Electrophoresis conditions: Use reducing conditions as demonstrated in published protocols

• Membrane transfer: PVDF membranes have been successfully used for TFEB detection

• Blocking and antibody incubation: Dilute primary antibody appropriately (1:1000-1:6000 for WB) , followed by compatible secondary antibody (e.g., HRP-conjugated Anti-Mouse IgG)

• Detection: Look for TFEB bands at approximately 65-70 kDa

• Buffers: For example, Immunoblot Buffer Group 1 has been used successfully with TFEB antibodies

What are the critical parameters for successful immunofluorescence staining of TFEB?

For successful immunofluorescence detection of TFEB:

• Fixation method: Immersion fixation has been documented for cell lines such as A549

• Antibody concentration: 3 μg/mL for 3 hours at room temperature has worked for certain applications

• Secondary antibody selection: Compatible fluorophore-conjugated secondary antibodies such as NorthernLights™ 557-conjugated Anti-Mouse IgG

• Counterstaining: DAPI can be used for nuclear visualization

• Expected localization: TFEB can show both cytoplasmic and nuclear localization depending on cellular state and stimulation conditions

• Cellular models: Successfully detected in multiple cell lines including HeLa and A549

• Controls: Include appropriate negative controls and positive controls where possible

How can I determine the optimal antibody dilution for my specific experiment?

Determining optimal antibody dilution requires systematic titration:

• Start with manufacturer's recommended range (e.g., 1:50-1:500 for IF/ICC, 1:1000-1:6000 for WB)

• Perform a dilution series experiment covering at least 3-4 dilutions (e.g., 1:500, 1:1000, 1:2000, 1:4000)

• Evaluate signal-to-noise ratio at each dilution

• Consider cell/tissue type specificity, as different samples may require different optimal dilutions

• Document conditions for reproducibility

• For specialized applications like ChIP, follow specific protocols (e.g., using 10 μl of antibody with 10 μg of chromatin)

How can TFEB antibodies be used to study TFEB phosphorylation states?

Studying TFEB phosphorylation requires specialized approaches:

• Phospho-specific antibodies: Researchers have developed antibodies targeting specific phosphorylation sites including p-S466, p-S467, and p-S469

• Validation approach: These antibodies should be validated using phospho-peptides to confirm site specificity

• Cell-free assays: Bacterially expressed proteins can be used to evaluate phosphorylation of TFEB C-terminal serine residues by kinases like AMPK

• Cross-reactivity assessment: Carefully evaluate whether phospho-specific antibodies cross-react with nearby phosphorylation sites (e.g., p-S466/S467 antibody may recognize dually phosphorylated p-S466/S467 and single p-S467 peptide, with lesser reactivity to single p-S466 peptide)

• Sensitivity considerations: Some phospho-antibodies (e.g., p-S469) may be less sensitive compared to others

How do I study TFEB nuclear translocation in response to cellular stimuli?

TFEB nuclear translocation studies require:

What considerations are important when using TFEB antibodies in ChIP experiments?

For successful ChIP experiments using TFEB antibodies:

• Antibody amount: Use 10 μl of antibody with 10 μg of chromatin (approximately 10^6 cells) per IP

• Validation: Use validated antibodies specifically tested for ChIP applications

• Protocol selection: Consider using optimized kits such as SimpleChIP® Enzymatic Chromatin IP Kits which have been validated with TFEB antibodies

• Controls: Include appropriate input controls, negative controls (IgG), and positive controls (known TFEB target genes)

• Cross-linking conditions: Optimize formaldehyde cross-linking time for your specific cell type

• Sonication parameters: Ensure chromatin is properly fragmented to appropriate size (typically 200-500 bp)

Why might I detect multiple bands or unexpected molecular weights when probing for TFEB?

Multiple bands or unexpected weights may occur due to:

• Post-translational modifications: TFEB has a calculated weight of 53 kDa but is typically observed at 65-70 kDa due to phosphorylation and other modifications

• Splice variants: Check for known TFEB isoforms that may be detected by your antibody

• Proteolytic cleavage: Sample degradation during preparation can generate fragments

• Non-specific binding: Some antibodies may cross-react with related transcription factors (especially other MiT/TFE family members)

• Sample preparation conditions: Reducing vs. non-reducing conditions may affect band pattern

• Antibody specificity: Verify the epitope recognized by your antibody; some target specific regions (e.g., Pro384-Ala446)

How can I verify the specificity of my TFEB antibody?

Antibody specificity verification methods include:

• Genetic controls: Use TFEB knockout or knockdown samples as negative controls

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple antibodies: Use different antibodies targeting distinct TFEB epitopes and compare results

• Recombinant protein: Compare detection of recombinant TFEB with endogenous detection

• Cross-species reactivity: Check whether observed reactivity matches predicted species reactivity (human, mouse, rat)

• Blocking peptides: If available, use specific blocking peptides to confirm binding specificity

What controls should I include when studying TFEB localization or expression?

Essential controls for TFEB localization or expression studies:

• Positive control tissues/cells: Use samples known to express TFEB (e.g., Raji cells, A549 cells, HeLa cells, Jurkat cells)

• Negative control: Secondary antibody only (omit primary antibody)

• Stimulus response controls: For nuclear translocation studies, include both unstimulated and stimulated samples

• Cell type controls: Compare TFEB expression/localization across different cell populations (e.g., naïve vs. memory B cells)

• Subcellular marker controls: Include nuclear markers (DAPI) and other organelle markers when studying localization

• Loading/housekeeping controls: For expression studies, include appropriate loading controls

How can TFEB antibodies be used to study the relationship between TFEB and autophagy regulation?

Studying TFEB's role in autophagy regulation requires:

• Stimulus selection: Choose treatments known to affect autophagy (starvation, mTOR inhibitors)

• Co-localization studies: Use TFEB antibodies alongside markers of lysosomes and autophagosomes

• Nuclear-cytoplasmic fractionation: Separate nuclear and cytoplasmic fractions to quantify TFEB translocation

• Autophagy markers: Combine TFEB detection with autophagy markers (LC3, p62)

• Signaling pathway analysis: Study TFEB in context of regulatory pathways (mTOR, AMPK)

• Genetic manipulation: Combine with TFEB overexpression or knockdown to establish causality

• Target gene expression: Measure expression of TFEB target genes involved in autophagy

The extensive literature on TFEB's role in autophagy (referenced in search results) provides background and experimental design ideas .

What methodological approaches can be used to study TFEB in B cell activation and immune response?

To investigate TFEB in B cell immunity:

• Cell isolation: Purify B cell populations (naïve, memory, plasmablasts) using CD markers (CD19+, IgD+/-, CD27+/-, CD38+/-)

• Stimulation protocols: Use BCR ligation techniques to activate B cells

• Flow cytometry: Measure TFEB expression across different B cell subsets

• Nuclear translocation: Quantify nuclear vs. cytoplasmic TFEB in response to stimulation

• Functional assays: Correlate TFEB activity with B cell functions (proliferation, antibody production)

• Co-stimulatory signals: Investigate how CD80 expression correlates with TFEB activation

• Cross-species comparison: Compare TFEB behavior in B cells across species (mouse vs. human)

Recent research has established TFEB as a "cross-isotype BCR-distal nuclear effector" and "inter-species marker of B cells with antigenic experience" .

This table summarizes findings from recent research on TFEB in human B cell subsets .

How can I integrate TFEB antibody data with other omics approaches?

Integrating TFEB research with multi-omics requires:

• Immunoprecipitation-mass spectrometry: Use TFEB antibodies for IP followed by MS to identify interacting partners (approach mentioned in research using TFEB-GFP KI MEF cells)

• ChIP-seq: Use ChIP-validated TFEB antibodies to identify genome-wide binding sites

• Proteomics correlation: Compare TFEB protein levels (antibody detection) with global proteome changes

• Transcriptomics integration: Correlate TFEB nuclear localization with expression of known TFEB target genes

• Phospho-proteomics: Use phospho-specific TFEB antibodies alongside global phosphorylation profiling

• Systems biology approaches: Model TFEB activity in context of broader cellular networks

• Multi-parametric imaging: Combine TFEB antibody staining with other markers for high-content analysis

What experimental design considerations are important when studying TFEB phosphorylation by AMPK?

When investigating TFEB phosphorylation by AMPK:

• Phospho-specific antibodies: Use antibodies targeting specific phosphorylation sites (p-S466, p-S467, p-S469)

• Kinase assays: Perform in vitro kinase assays using purified components

• Cellular activation: Use AMPK activators and inhibitors to modulate the pathway

• Mutational analysis: Create phospho-mimetic or phospho-dead TFEB mutants

• Sequential phosphorylation: Consider whether phosphorylation at one site influences modification at adjacent sites

• Species differences: Account for sequence differences between human and mouse TFEB when designing experiments

• Pathway integration: Consider the interplay between AMPK and other kinases (mTOR) in regulating TFEB

Recent research has investigated how "AMPK activation promotes transcriptional activation of TFEB" , providing experimental frameworks for these studies.

How can researchers address discrepancies in TFEB antibody reactivity across different experimental systems?

To address discrepancies in antibody performance:

• Validation across multiple systems: Test antibodies in different cell types and tissues to establish reliability

• Multiple detection methods: Compare results from different techniques (WB, IF, IHC, flow cytometry)

• Epitope mapping: Understand exactly which region of TFEB your antibody recognizes

• Post-translational modifications: Consider how modifications might affect epitope accessibility

• Sample preparation variations: Standardize protocols for sample preparation

• Antibody combinations: Use multiple antibodies targeting different epitopes

• Quantitative standards: Include recombinant TFEB standards for quantitative comparisons