TMF1 Antibody

What is TMF1 and what cellular functions make it a significant target for antibody-based research?

TMF1 (TATA element modulatory factor 1), also known as ARA160, is a multifunctional protein with several important cellular roles. Understanding these functions is essential for designing experiments with TMF1 antibodies:

• Serves as a potential coactivator of the androgen receptor

• Mediates STAT3 degradation

• Plays critical roles in two RAB6-dependent retrograde transport processes: from endosomes to the Golgi and from the Golgi to the ER

• Binds the HIV-1 TATA element and inhibits transcriptional activation by the TATA-binding protein (TBP)

The protein has a calculated molecular weight of 123 kDa but is typically observed at 150-160 kDa in Western blots due to post-translational modifications . TMF1 is widely expressed across multiple tissue types, making it relevant for various research applications .

What are the optimal experimental conditions for TMF1 antibody applications in Western blotting?

When performing Western blot experiments with TMF1 antibodies, the following parameters should be considered for optimal results:

Recommended dilutions:

• Polyclonal antibodies: 1:500-1:2000

• Monoclonal antibodies: 1:5000-1:50000

Validated cell lines for positive detection:

• Human cell lines: BxPC-3, L02, HeLa, HepG2, HEK-293, Jurkat, K-562

• Rodent cell lines: HSC-T6, NIH/3T3

Expected molecular weight: 150-160 kDa (although calculated MW is 123 kDa)

Methodological considerations:

• Use freshly prepared cell lysates

• Include positive control lysates from validated cell lines

• Validate antibody specificity using appropriate blocking peptides

• Consider nonspecific binding patterns when interpreting results

How should samples be prepared for optimal TMF1 detection in immunohistochemistry (IHC)?

For successful IHC detection of TMF1, sample preparation is critical:

Antigen retrieval recommendations:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Validated tissue samples:

• Human cervical cancer tissue

• Human testis tissue

• Mouse brain tissue

• Mouse kidney tissue

• Rat brain tissue

Recommended dilution range: 1:50-1:500 for polyclonal antibodies; 1:250-1:1000 for monoclonal antibodies

Methodological protocol:

• Fix tissue samples in 10% neutral buffered formalin

• Embed in paraffin and section at 4-6 μm

• Perform antigen retrieval as specified above

• Block endogenous peroxidase and nonspecific binding sites

• Incubate with primary antibody at optimal dilution

• Detect using appropriate secondary antibody and visualization system

• Counterstain, dehydrate, and mount

What troubleshooting approaches should be used when encountering weak or no signal in ELISA experiments with TMF1 antibodies?

When facing signal problems in ELISA with TMF1 antibodies, consider these systematic troubleshooting steps:

For no signal or weak signal:

For high background:

How can researchers effectively validate the specificity of TMF1 antibodies?

Antibody validation is crucial for ensuring experimental reliability. For TMF1 antibodies, a comprehensive validation approach should include:

Multiple validation methods:

• Western blot analysis: Confirm single band at expected molecular weight (150-160 kDa) in validated cell lysates (HepG2, HeLa)

• Immunoprecipitation: Validate using HepG2 cells (0.5-4.0 μg antibody for 1.0-3.0 mg total protein)

• Immunostaining pattern analysis: Compare subcellular localization with known TMF1 distribution (Golgi, ER, nucleus)

• Knockout/knockdown controls: Compare staining in TMF1-depleted versus wild-type samples

• Cross-reactivity testing: Test across multiple species if claiming multi-species reactivity

Important considerations:

• Avoid using only recombinant proteins for validation as this provides limited specificity information

• Use cell lysates rather than purified proteins to evaluate cross-reactivity

• Document observed molecular weight compared to calculated weight (123 kDa vs. 150-160 kDa)

What are the critical differences between monoclonal and polyclonal TMF1 antibodies for specific research applications?

Understanding the differences between monoclonal and polyclonal TMF1 antibodies is essential for experimental design:

Polyclonal TMF1 antibodies:

• Host: Typically rabbit

• Dilution ranges: 1:500-1:2000 (WB); 1:50-1:500 (IHC); 1:50-1:500 (IF/ICC)

• Advantages: Recognize multiple epitopes; generally higher sensitivity; better for detecting denatured proteins

• Applications: Effective across WB, IHC, IF/ICC, IP, ELISA

Monoclonal TMF1 antibodies:

• Host: Typically mouse

• Dilution ranges: 1:5000-1:50000 (WB); 1:250-1:1000 (IHC); 1:400-1:1600 (IF/ICC)

• Advantages: Highly consistent lot-to-lot; specific to single epitope; lower background in some applications

• Applications: Particularly effective for WB and quantitative applications

Selection guidance:

• Choose polyclonal for exploratory research or when protein may be modified/degraded

• Choose monoclonal for consistent, reproducible results and quantitative analysis

• Consider using both antibody types to validate observations, especially in novel research areas

How can TMF1 antibodies be effectively used to study protein-protein interactions in cellular transport processes?

TMF1 plays crucial roles in RAB6-dependent retrograde transport processes, making it an interesting target for studying protein trafficking interactions:

Recommended methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use TMF1 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein)

  • Validated cell lines: HepG2 cells

  • Analyze precipitates for known interacting proteins (RAB6, androgen receptor, STAT3)

• Proximity Ligation Assay (PLA):

  • Use TMF1 antibodies in combination with antibodies against suspected interacting proteins

  • Optimal dilution for IF/ICC applications: 1:50-1:500 (polyclonal) or 1:400-1:1600 (monoclonal)

  • Analyze dots indicating protein-protein interactions in situ

• Immunofluorescence co-localization:

  • Use TMF1 antibodies (1:50-1:500) together with markers for cellular compartments

  • Validated cell lines: HepG2, A431 cells

  • Assess co-localization with Golgi, ER markers, or transport proteins

Experimental considerations:

• Preserve protein-protein interactions by using mild lysis conditions

• Include appropriate controls to validate interactions

• Consider dynamic nature of transport processes when designing temporal studies

What role do anti-TIF1-γ (anti-transcriptional intermediary factor 1 gamma) antibodies play in cancer and dermatomyositis research?

Anti-TIF1-γ antibodies are important biomarkers in clinical research, particularly in the context of dermatomyositis (DM) and cancer:

Cancer association findings:

• In 88% of anti-TIF1-γ positive patients, cancer was diagnosed within 3 years from DM diagnosis compared to 63% in anti-TIF1-γ negative patients

• Anti-TIF1-γ antibodies can be detected up to five years before cancer diagnosis in some patients

• Higher antibody levels (median 2.13 au) were associated with mortality within 1 year after cancer diagnosis compared to lower levels (median 1.34 au) in patients surviving longer

• Remission of cancer often coincides with DM remission and decrease in anti-TIF1-γ antibody levels

Detection methods compared:

*CADM = Cancer-Associated Dermatomyositis

Methodological recommendations:

• For research applications: Use multiple detection methods for validation

• For clinical applications: Consider ELISA for screening due to higher sensitivity, with IP confirmation for borderline cases

• Monitor antibody levels longitudinally in studies of cancer progression/remission

What considerations should be made when developing ELISA assays for TMF1 antibody detection?

When developing an ELISA for TMF1 antibody detection, researchers should consider these key parameters:

Assay design considerations:

• Antigen coating:

  • Optimal concentration of recombinant TMF1: ~0.25 μg/ml in carbonate buffer (pH 9.6)

  • Coating temperature and time: 4°C overnight

• Plate selection:

  • Use high-binding capacity plates specifically validated for ELISA

  • Avoid tissue culture plates

• Sample handling:

  • Run samples in duplicate to control for technical variation

  • Include standard curves on every plate to account for inter-plate variation

  • Consider sample dilution series to ensure readings within linear range

• Quality control measures:

  • Include known positive and negative controls

  • Incorporate internal standard samples across plates

  • Evaluate intra- and inter-assay coefficients of variation

Troubleshooting poor dynamic range:

How can antibody developability assessments improve TMF1 antibody research reliability?

Antibody developability studies can significantly enhance experimental reproducibility. For TMF1 antibodies, consider these approaches:

Key developability parameters to assess:

• Stability testing:

  • Short-term stability at 2-8°C, 25°C, and 40°C

  • Freeze-thaw stability (important for TMF1 antibodies stored with 50% glycerol)

  • Limited forced degradation studies

• Specificity evaluations:

  • Cross-reactivity profiling across multiple cell lines

  • Species reactivity testing (human, mouse, rat)

  • Epitope mapping to identify binding regions

• Performance characteristics:

  • Assessment across multiple applications (WB, IHC, IF/ICC, IP, ELISA)

  • Dilution optimization for each application

  • Comparison with reference antibodies

Biophysical property assessments:

• Thermal stability measurements

• Colloidal stability evaluations

• Hydrophobicity and charge characteristics

Research shows that antibodies with fewer developability "flags" have greater success in downstream applications, highlighting the importance of comprehensive characterization before extensive use in research programs .

What methodologies can be used to investigate TMF1's role in androgen receptor signaling?

As a potential coactivator of the androgen receptor, TMF1 represents an important target for research into steroid hormone signaling:

Recommended experimental approaches:

• Co-immunoprecipitation studies:

  • Use TMF1 antibodies at 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Ideal cell models: LNCaP cells (androgen-responsive)

  • Analyze for presence of androgen receptor in precipitates

• Chromatin immunoprecipitation (ChIP):

  • Use TMF1 antibodies to investigate recruitment to androgen response elements

  • Compare occupancy patterns with and without androgen stimulation

  • Consider sequential ChIP to identify co-recruitment with androgen receptor

• Functional studies:

  • Combine TMF1 antibodies with androgen receptor activity reporters

  • Use immunofluorescence (IF) at dilutions of 1:50-1:500 to examine co-localization

  • Monitor changes in localization and expression following androgen treatment

Experimental considerations:

• Include appropriate hormone treatments (e.g., DHT) and controls (e.g., flutamide)

• Account for cell cycle dependencies in androgen signaling

• Consider nuclear vs. cytoplasmic fractionation in biochemical analyses

How can researchers effectively troubleshoot variability in TMF1 antibody experiments?

Experimental variability is a common challenge in antibody-based research. For TMF1 antibodies, consider these strategies:

Sources of variability and solutions:

Methodological recommendations:

• Implement rigorous protocols with detailed SOPs

• Consider using automation where possible to reduce human error

• Run technical replicates (minimum of duplicates)

• Include positive controls from validated cell lines (HepG2, HeLa)

• Maintain consistent antibody storage conditions (-20°C, avoid repeated freeze/thaw)

What are advanced applications of TMF1 antibodies in studying retrograde transport mechanisms?

TMF1's involvement in RAB6-dependent retrograde transport processes offers opportunities for studying cellular trafficking mechanisms:

Advanced methodological approaches:

• Live-cell imaging:

  • Combine TMF1 antibody fragments with cell-penetrating peptides

  • Track movement of TMF1-containing complexes in real time

  • Correlate with RAB6 dynamics using dual-color imaging

• Super-resolution microscopy:

  • Use TMF1 antibodies at optimized dilutions (1:50-1:500 for IF/ICC)

  • Validated in HepG2 and A431 cells

  • Employ multi-color STORM or STED to visualize TMF1 in relation to Golgi and ER markers

• Proximity-dependent labeling:

  • Combine TMF1 antibodies with BioID or APEX2 approaches

  • Identify novel proteins involved in TMF1-mediated transport

  • Map the dynamic TMF1 interactome during transport events

Experimental design considerations:

• Include appropriate transport inhibitors as controls (e.g., Brefeldin A)

• Consider temporal dynamics in experimental design

• Use multiple complementary approaches to validate findings

How should researchers approach epitope mapping for TMF1 antibodies?

Epitope mapping provides crucial information about antibody binding characteristics and can explain cross-reactivity patterns:

Methodological approaches for TMF1 epitope mapping:

• Peptide array analysis:

  • Generate overlapping peptide fragments spanning the 1093 amino acid sequence of TMF1

  • Test antibody binding to identify specific epitope regions

  • Compare to known functional domains of TMF1

• Deletion and mutation analysis:

  • Create truncated or mutated TMF1 constructs

  • Assess antibody binding via Western blot or ELISA

  • Identify critical residues for antibody recognition

• Competition assays:

  • Use defined peptides to compete for antibody binding

  • Measure inhibition of binding to full-length TMF1

  • Determine relative affinities for different epitopes

Practical considerations:

• Account for potential conformational epitopes not represented in linear peptides

• Consider post-translational modifications that might affect epitope recognition

• Use epitope information to predict potential cross-reactivity with related TATA modulatory factors

• Apply findings to understand differences between observed (150-160 kDa) and calculated (123 kDa) molecular weights

What are the latest developments in using TMF1 antibodies for research into viral interactions?

TMF1 binds the HIV-1 TATA element and inhibits transcriptional activation by TBP, making it relevant for viral research:

Emerging research approaches:

• Virus-host protein interaction studies:

  • Use TMF1 antibodies in immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg protein)

  • Analyze precipitates for viral proteins or viral DNA/RNA

  • Investigate changes in TMF1 localization during viral infection

• Transcriptional regulation analysis:

  • Combine ChIP assays using TMF1 antibodies with analysis of viral promoters

  • Compare TMF1 occupancy at viral versus cellular TATA elements

  • Correlate with transcriptional output using reporter assays

• Connection to antiviral immunity:

  • Research suggests connections between TMF1 and antiviral responses

  • Antibodies against TRIM family proteins (including TMF1) show associations with viral epitopes

  • This pattern may extend to poxviruses and other viral families

Research considerations:

• Include appropriate viral infection models and controls

• Consider temporal dynamics of viral replication cycle

• Account for potential TMF1 post-translational modifications during infection

Human's query was about generating FAQs for TMF1 antibody research, and I've provided 15 comprehensive FAQs covering basic to advanced research aspects, with methodological answers and data tables as requested.