TFB1M Antibody, Biotin conjugated

What is TFB1M and why is it significant in mitochondrial research?

TFB1M (Transcription Factor B1, Mitochondrial), also known as Dimethyladenosine transferase 1, plays a critical role in mitochondrial gene expression. The protein is essential for proper transcription of mitochondrial DNA, making it a key focus in studies of mitochondrial biogenesis and function. Research has established that the expression of human TFB1M is governed by nuclear respiratory factors (NRF-1 and NRF-2), which are key transcription factors in mitochondrial biogenesis pathways . TFB1M works in coordination with other factors such as Tfam and is up-regulated in cellular systems where mitochondrial biogenesis is induced, particularly in response to PGC-1α or PRC coactivator activity .

What types of biotin-conjugated TFB1M antibodies are available for research?

Based on available research materials, there are several biotin-conjugated TFB1M antibodies with different properties:

The polyclonal antibody is generated against recombinant Human Dimethyladenosine transferase 1, mitochondrial protein (59-194AA) , while the monoclonal antibody is raised against a human recombinant protein fragment corresponding to amino acids 116-346 of human TFB1M (NP_057104) .

How do biotin-conjugated antibodies differ from unconjugated versions?

Biotin conjugation provides several advantages over unconjugated antibodies:

• Enhanced detection sensitivity through streptavidin-based amplification systems

• Increased flexibility in experimental design, allowing for multiple detection strategies

• Compatibility with a wide range of streptavidin-conjugated reporter molecules

• Reduced background in certain applications due to the high specificity of biotin-streptavidin interaction

• Ability to be used in complex multi-step staining protocols where direct conjugation might interfere with epitope recognition

The biotin conjugation does not appear to significantly alter the binding specificity of the antibodies, as they maintain their reactivity to the target TFB1M protein .

What are the validated applications for biotin-conjugated TFB1M antibodies?

The biotin-conjugated TFB1M antibodies have been validated for different applications depending on the specific antibody:

• ELISA applications: The rabbit polyclonal antibody (AA 59-194) has been specifically validated for ELISA techniques .

• Western Blot applications: The mouse monoclonal antibody (OTI11H1) has been validated for Western Blot with a recommended dilution of 1:2000 . When used in Western Blot, the antibody can detect TFB1M protein with a predicted size of 39.4 kDa .

• Potential for additional applications: While not specifically validated for biotin-conjugated versions, other TFB1M antibodies have been used successfully in immunohistochemistry (IHC) and immunofluorescence (IF), suggesting that with proper optimization, biotin-conjugated versions may also work for these applications .

What is the optimal protocol for Western Blot using biotin-conjugated TFB1M antibody?

For optimal Western Blot results using the biotin-conjugated TFB1M monoclonal antibody (OTI11H1):

• Sample preparation: Load approximately 25 μg of protein extract per lane on a 12% SDS-PAGE gel .

• Transfer: Transfer proteins to a nitrocellulose or PVDF membrane using standard techniques.

• Blocking: Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute the biotin-conjugated TFB1M antibody 1:2000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash the membrane 3-5 times with TBST.

• Detection: Incubate with a streptavidin-HRP conjugate at the appropriate dilution.

• Development: Visualize using enhanced chemiluminescence (ECL) or other compatible detection methods.

Note that the antibody has been purified from mouse ascites fluids or tissue culture supernatant by affinity chromatography (protein A/G), ensuring high specificity .

How should biotin-conjugated TFB1M antibodies be stored to maintain activity?

For optimal storage and handling of biotin-conjugated TFB1M antibodies:

• Long-term storage: Store at -20°C as received in small aliquots to prevent freeze-thaw cycles .

• Short-term storage (up to 1 week): Store at 2-8°C .

• Working solutions: Prepare fresh dilutions on the day of use.

• Buffer conditions: The monoclonal antibody is supplied in PBS (pH 7.3) containing 1% BSA, 50% glycerol, and 0.02% sodium azide .

• Stability: When properly stored, the antibody is stable for 12 months from the date of receipt .

Proper storage is critical as repeated freeze-thaw cycles can lead to denaturation and loss of binding activity, particularly for biotin-conjugated antibodies which may be more sensitive to storage conditions.

How can biotin-conjugated TFB1M antibodies be used to study mitochondrial biogenesis pathways?

Biotin-conjugated TFB1M antibodies can be powerful tools for investigating mitochondrial biogenesis:

• Expression analysis: Quantify TFB1M protein levels in response to stimuli known to induce mitochondrial biogenesis, such as exercise, caloric restriction, or cold exposure.

• Co-regulation studies: Examine the coordinated expression of TFB1M with other mitochondrial transcription factors such as Tfam and TFB2M during biogenesis .

• Transcriptional control: Investigate the relationship between TFB1M expression and its regulators, particularly the NRF-1 and NRF-2 transcription factors and PGC-1 family coactivators .

• Protein interactions: Use in co-immunoprecipitation experiments to identify protein complexes containing TFB1M.

• Chromatin studies: Employ in ChIP assays to examine TFB1M binding to mitochondrial DNA and its role in transcription initiation.

The biotin conjugation provides additional flexibility in these applications by enabling various detection strategies without requiring direct enzyme conjugation to the primary antibody.

What controls should be included when using biotin-conjugated TFB1M antibodies?

For rigorous experimental validation with biotin-conjugated TFB1M antibodies, the following controls are essential:

• Positive control: Include cell or tissue lysates known to express TFB1M, such as 721_B cells as indicated by BioGPS gene expression data .

• Negative control: Utilize samples from TFB1M knockout models or cell lines with confirmed low expression.

• Blocking peptide control: Pre-incubate the antibody with the immunizing peptide to confirm specificity. For the middle region antibody, the blocking peptide sequence is: IIKWLENISCRDGPFVYGRTQMTLTFQKEVAERLAANTGSKQRSRLSVMA .

• Isotype control: Include an irrelevant biotin-conjugated antibody of the same isotype (IgG for polyclonal, IgG1 for the monoclonal) to control for non-specific binding.

• Endogenous biotin control: Since many tissues (particularly liver, kidney, and brain) contain endogenous biotin, include a control without primary antibody but with streptavidin detection to assess background.

• Loading control: Always include appropriate loading controls when conducting Western blots to normalize TFB1M expression levels.

How can TFB1M expression be correlated with changes in mitochondrial function?

To establish meaningful correlations between TFB1M expression and mitochondrial function:

• Parallel measurements: Simultaneously assess TFB1M protein levels using the biotin-conjugated antibodies alongside mitochondrial functional parameters such as oxygen consumption, ATP production, and membrane potential.

• Intervention studies: Examine how manipulations that alter mitochondrial function (e.g., respiratory chain inhibitors, uncouplers) affect TFB1M expression.

• Genetic approaches: Compare TFB1M levels in models with genetic alterations in mitochondrial components.

• Longitudinal analysis: Track changes in TFB1M expression during processes known to affect mitochondrial biogenesis, such as differentiation or adaptation to metabolic challenges.

• Subcellular fractionation: Use the antibodies to determine the distribution of TFB1M between different cellular compartments and correlate with mitochondrial metrics.

This approach can help establish whether TFB1M serves as a reliable biomarker for mitochondrial biogenesis and function in various physiological and pathological contexts.

What are common issues when using biotin-conjugated TFB1M antibodies and how can they be resolved?

Several technical challenges may arise when working with biotin-conjugated TFB1M antibodies:

For Western blot applications specifically, loading 25 μg of protein extract and using the antibody at a 1:2000 dilution has been demonstrated to produce reliable results .

How can the specificity of biotin-conjugated TFB1M antibodies be verified?

Verifying antibody specificity is critical for reliable research outcomes:

• Peptide competition: Pre-incubate the antibody with excess immunizing peptide before application to confirm that signal loss occurs with specific binding.

• Genetic validation: Compare staining patterns or band intensity in samples with TFB1M overexpression versus knockdown/knockout.

• Size verification: Confirm that the detected protein matches the predicted molecular weight of TFB1M (39.4 kDa) .

• Cross-species validation: Test the antibody in samples from multiple species where cross-reactivity is predicted based on sequence homology. The polyclonal middle-region antibody has predicted homology of: Cow: 86%; Dog: 93%; Guinea Pig: 86%; Horse: 86%; Human: 100%; Mouse: 86%; Rabbit: 93%; Rat: 86%; Zebrafish: 79% .

• Orthogonal methods: Correlate antibody results with mRNA expression data or mass spectrometry protein identification.

What modifications can be made to standard protocols for specialized applications of biotin-conjugated TFB1M antibodies?

When adapting biotin-conjugated TFB1M antibodies for specialized applications:

• For co-localization studies:

  • Use low antibody concentrations to minimize background

  • Consider tyramide signal amplification for enhanced sensitivity

  • Optimize blocking to prevent non-specific streptavidin binding

• For flow cytometry:

  • Increase permeabilization time to ensure antibody access to mitochondrial proteins

  • Use appropriate compensation controls for multiple fluorophores

  • Consider fixation methods that preserve mitochondrial structure

• For proximity ligation assays:

  • Pair with antibodies against potential interaction partners

  • Optimize fixation to preserve protein-protein interactions

  • Reduce background by extensive washing

• For super-resolution microscopy:

  • Use small streptavidin-conjugated fluorophores for better resolution

  • Consider photoconvertible streptavidin conjugates for techniques like PALM

  • Minimize fixation-induced epitope masking

• For multiplexed detection:

  • Employ biotin-conjugated TFB1M antibody as part of a sequential staining protocol

  • Use streptavidin conjugates with spectrally distinct fluorophores for parallel detection

  • Consider antibody stripping and reprobing strategies for multiple targets

How can biotin-conjugated TFB1M antibodies be used to study mitochondrial dysfunction in disease?

Biotin-conjugated TFB1M antibodies offer valuable approaches for investigating mitochondrial dysfunction in various diseases:

• Comparative expression analysis: Quantify TFB1M protein levels in affected versus normal tissues to identify disease-associated changes in mitochondrial transcription machinery.

• Therapeutic response monitoring: Track changes in TFB1M expression during treatment with compounds targeting mitochondrial function.

• Disease progression studies: Examine TFB1M levels at different stages of disease development to establish temporal relationships with mitochondrial dysfunction.

• Cellular stress response: Investigate how cellular stressors associated with disease states affect TFB1M expression and localization.

• Biomarker development: Evaluate whether TFB1M protein levels correlate with disease severity or progression, potentially serving as a biomarker for mitochondrial involvement.

The biotin conjugation provides flexibility in detection methods, particularly valuable when working with limited or precious clinical samples.

What is known about TFB1M regulation by nuclear respiratory factors and how can this be studied?

The regulation of TFB1M by nuclear respiratory factors (NRFs) represents a key mechanism in mitochondrial biogenesis:

• Promoter structure: The hTFB1M promoter (accession number AL139101) contains binding sites for NRF-1 and NRF-2, which can be studied through promoter deletion and site-directed mutagenesis approaches .

• Transcriptional regulation: NRF-1 and NRF-2 bind to specific sequences in the TFB1M promoter to drive its expression, with the NRF-1 binding site located within the region corresponding to nucleotides -143 to -117 of the promoter .

• Coactivator involvement: PGC-1α and PRC function as coactivators for NRFs in driving TFB1M expression, establishing a regulatory network that coordinates nuclear and mitochondrial gene expression .

• Coordinate regulation: TFB1M is up-regulated along with other mitochondrial transcription factors (Tfam, TFB2M) in response to signals that induce mitochondrial biogenesis .

Biotin-conjugated TFB1M antibodies can be used to investigate these regulatory mechanisms by:

• Quantifying TFB1M protein levels after manipulation of NRF expression or activity

• Examining the kinetics of TFB1M protein expression following activation of the PGC-1α pathway

• Comparing TFB1M protein levels across tissues with different metabolic demands and NRF activity

How can multiplexing with biotin-conjugated TFB1M antibodies enhance mitochondrial research?

Multiplexing approaches using biotin-conjugated TFB1M antibodies can significantly enhance mitochondrial research:

• Co-localization studies: Combine biotin-conjugated TFB1M antibody (detected with streptavidin-fluorophore conjugates) with directly labeled antibodies against other mitochondrial proteins to examine spatial relationships within the organelle.

• Protein complex analysis: Use in conjunction with antibodies against potential interaction partners to identify novel TFB1M-containing complexes.

• Pathway activation assessment: Simultaneously examine TFB1M expression alongside markers of mitochondrial biogenesis pathways (e.g., phosphorylated AMPK, nuclear PGC-1α) to establish regulatory relationships.

• Dynamic process monitoring: Combine with indicators of mitochondrial function (membrane potential dyes, superoxide indicators) to correlate TFB1M expression with functional outcomes.

• Tissue heterogeneity analysis: In tissue sections, use with cell-type-specific markers to examine cell-specific variation in TFB1M expression.

The versatility of biotin-streptavidin detection systems allows for signal amplification and flexible experimental design in these multiplexed approaches.