TBRG1 Antibody

What is TBRG1 and what are its key cellular functions?

TBRG1 (Transforming Growth Factor Beta Regulator 1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers. It gained its name from transcriptional regulation by TGF-β. TBRG1 can activate p53/TP53, cause G1 arrest, and collaborate with CDKN2A to restrict proliferation, but does not require either protein to inhibit DNA synthesis. It redistributes CDKN2A into the nucleoplasm and is involved in maintaining chromosomal stability . Recent studies have also revealed TBRG1's involvement in innate immune responses in invertebrates, suggesting evolutionary conservation of certain functions beyond growth regulation .

What types of TBRG1 antibodies are currently available for research applications?

Several types of TBRG1 antibodies are available for research applications, primarily polyclonal rabbit antibodies. These include:

Most commercially available antibodies are validated for Western blot analysis, while some are also validated for immunohistochemistry on paraffin-embedded tissues and ELISA applications .

How should TBRG1 antibodies be stored and handled to maintain optimal activity?

TBRG1 antibodies should be stored at -20°C for long-term stability. For products containing glycerol (typically 50%), aliquoting is not necessary for -20°C storage, but for antibodies without glycerol, aliquoting is recommended to avoid freeze-thaw cycles that can compromise antibody activity . Most antibodies are supplied in PBS with preservatives such as 0.02% sodium azide and may contain stabilizers such as glycerol (50%) or sucrose (2%) . Working dilutions should be prepared freshly before use. When preparing antibody dilutions, researchers should use proper buffers (typically PBS with 0.1-0.5% BSA or casein) and avoid contamination .

What are the recommended protocols for using TBRG1 antibodies in Western blot analysis?

For Western blot using TBRG1 antibodies, follow these methodological steps:

• Sample preparation: Prepare cell/tissue lysates using RIPA buffer supplemented with protease inhibitors

• Protein quantification: Use Bradford or BCA assay to normalize loading (15-30 μg total protein recommended)

• Gel electrophoresis: Separate proteins on 10-12% SDS-PAGE gels

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes

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

• Primary antibody incubation: Dilute TBRG1 antibody at recommended ratios (typically 1:500-1:2000) in blocking buffer and incubate overnight at 4°C

• Washing: Wash membranes 3-5 times with TBST

• Secondary antibody incubation: Use appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG at 1:5000-1:10000) for 1 hour at room temperature

• Detection: Develop using ECL substrate and imaging systems

The expected molecular weight for TBRG1 is approximately 45 kDa, though theoretical calculations may show values around 29 kDa .

How can TBRG1 antibodies be used effectively in immunohistochemistry applications?

For effective immunohistochemistry with TBRG1 antibodies:

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin

• Sectioning: Cut 4-6 μm sections and mount on positively charged slides

• Deparaffinization and rehydration: Use xylene and graded alcohols

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Endogenous peroxidase blocking: Block with 3% hydrogen peroxide for 10 minutes

• Protein blocking: Block with 5% normal serum for 1 hour

• Primary antibody incubation: Dilute TBRG1 antibody at 1:200 (or as recommended) and incubate overnight at 4°C

• Detection system: Use appropriate detection system (e.g., HRP-polymer with DAB substrate)

• Counterstaining: Counterstain with hematoxylin

• Dehydration and mounting: Dehydrate through graded alcohols and mount

Validated results show successful staining in rat pancreatic tissue at 1:200 dilution with strong nuclear and moderate cytoplasmic staining patterns .

What validation methods should be employed to confirm TBRG1 antibody specificity?

For rigorous validation of TBRG1 antibody specificity, employ the following strategies based on enhanced validation principles:

• Orthogonal validation: Compare protein levels determined by antibody-dependent methods (Western blot) with antibody-independent methods (mass spectrometry or RNA-seq) across multiple cell lines. Correlation coefficient >0.5 indicates validation .

• Genetic knockdown validation: Perform siRNA knockdown of TBRG1 in appropriate cell lines (e.g., U-2 OS) and demonstrate reduced signal in Western blot or immunostaining compared to control siRNA .

• Recombinant expression validation: Overexpress tagged TBRG1 in cell lines and confirm increased signal detection at the expected molecular weight .

• Independent antibody validation: Use multiple antibodies targeting different epitopes of TBRG1 and confirm similar staining patterns .

• Capture mass spectrometry: Perform immunoprecipitation with the TBRG1 antibody followed by mass spectrometry to confirm target capture .

Ideally, at least two of these enhanced validation methods should be used to confirm antibody specificity before using in critical experiments .

How can TBRG1 antibodies be used to investigate its role in tumor suppression and cell cycle regulation?

To investigate TBRG1's role in tumor suppression and cell cycle regulation:

• Cell cycle analysis: Use TBRG1 antibodies in conjunction with cell cycle markers (cyclin D, cyclin E, p21) in immunofluorescence or flow cytometry to determine correlation with cell cycle phases.

• Co-immunoprecipitation: Employ TBRG1 antibodies to pull down protein complexes to identify interactions with p53, CDKN2A, and other cell cycle regulators.

• Chromatin immunoprecipitation (ChIP): Use TBRG1 antibodies in ChIP assays to identify genomic binding sites and potential transcriptional targets.

• Cancer cell line panels: Analyze TBRG1 expression across cancer cell line panels using validated antibodies to correlate with growth rates and tumorigenic potential.

• In vivo tumor models: Utilize TBRG1 antibodies for immunohistochemical analysis of tumor tissues from xenograft models with TBRG1 modulation.

These approaches can help elucidate how TBRG1 activates p53, causes G1 arrest, and maintains chromosomal stability in the context of tumor suppression .

What are the methodological considerations when using TBRG1 antibodies to study its role in immune responses?

Recent research has identified TBRG1's involvement in innate immunity in invertebrates . When studying its immune functions, consider:

• Cross-species reactivity: While studying TBRG1 in different species (e.g., Procambarus clarkii), assess antibody cross-reactivity through Western blot validation.

• Immune challenge models: Design experiments with appropriate immune challenges (bacterial, viral) and use TBRG1 antibodies to track expression changes by immunoblotting or immunohistochemistry.

• Combined RNAi and antibody approaches: Pair knockdown experiments with antibody detection to verify target reduction and correlate with phenotypic changes in immune responses.

• Antimicrobial peptide (AMP) expression analysis: Use TBRG1 antibodies alongside AMP antibodies to study potential regulatory relationships through co-localization studies.

• Bacteria clearance assays: Design phagocytosis assays using labeled bacteria and track TBRG1 localization during bacterial clearance using immunofluorescence microscopy.

These methodologies can help establish connections between TBRG1 expression and innate immune functions, particularly in understanding evolutionary conservation of these pathways .

How can TBRG1 antibodies be integrated into multi-omics approaches to understand its regulatory networks?

For integration of TBRG1 antibodies into multi-omics experimental designs:

• Proteogenomic correlation: Combine TBRG1 antibody-based protein quantification with RNA-seq data across cell line panels to identify post-transcriptional regulation mechanisms.

• Phospho-proteomics integration: Use TBRG1 immunoprecipitation followed by mass spectrometry to identify phosphorylation status and potential regulatory modifications.

• ChIP-seq and protein expression correlation: Correlate TBRG1 chromatin binding sites (ChIP-seq) with protein expression changes (antibody-based detection methods) to identify direct transcriptional targets.

• Protein interactome analysis: Use TBRG1 antibodies for immunoprecipitation followed by mass spectrometry to identify protein interaction networks in different cellular contexts.

• Single-cell multi-parameter analysis: Combine TBRG1 antibody staining with other markers in single-cell analysis platforms to identify cell type-specific expression patterns and correlations.

This multi-omics integration can reveal how TBRG1 functions within larger regulatory networks influencing cell growth, genomic stability, and immune responses .

What are common sources of non-specific binding with TBRG1 antibodies and how can they be mitigated?

Common sources of non-specific binding and their solutions include:

• Insufficient blocking: Increase blocking time (2-3 hours) or concentration (5-10% BSA/milk) and ensure complete coverage of membrane/slides.

• Cross-reactivity with similar epitopes: Perform peptide competition assays using the immunogen peptide to confirm specificity. Consider pre-absorption with the immunizing peptide.

• Secondary antibody background: Include secondary-only controls and consider using secondary antibodies specifically validated for low background.

• Sample preparation issues: Ensure complete cell lysis and protein denaturation for Western blot applications. For IHC, optimize fixation times and antigen retrieval methods.

• Antibody concentration too high: Perform titration experiments (1:200 to 1:2000) to determine optimal antibody concentration showing specific signal with minimal background .

• Buffer incompatibility: Ensure compatibility between sample buffer components and antibody formulation. Some buffer components (detergents, reducing agents) may affect antibody binding.

These optimization steps are particularly important when detecting TBRG1, as its expression levels can vary significantly across tissues and cell types.

How should researchers address discrepancies in molecular weight observations when using TBRG1 antibodies?

When addressing molecular weight discrepancies with TBRG1 antibodies:

• Expected vs. observed weight: TBRG1's theoretical molecular weight is reported as 29 kDa, but observed weight is often approximately 45 kDa . This discrepancy could be due to post-translational modifications or structural properties.

• Validation approach:

  • Run positive control samples with known TBRG1 expression

  • Include recombinant TBRG1 protein as a reference

  • Test multiple antibodies targeting different epitopes

  • Perform knockdown/knockout validation to confirm band identity

• Post-translational modifications: Consider that phosphorylation, glycosylation, or other modifications may alter migration patterns.

• Protein isoforms: Check databases for known TBRG1 isoforms that might explain different molecular weights.

• Denaturing conditions: Optimize SDS-PAGE conditions, including buffer compositions and reducing agent concentrations, to ensure complete denaturation.

• Resolution optimization: Use gradient gels (4-15%) to better resolve proteins in the 25-50 kDa range when trying to identify specific TBRG1 bands.

These approaches can help resolve discrepancies between theoretical and observed molecular weights, allowing for more accurate interpretation of Western blot results.

What strategies can be employed when TBRG1 antibodies show inconsistent results across different experimental platforms?

When facing inconsistent results across platforms:

• Application-specific validation: Validate antibodies specifically for each application (WB, IHC, IF) as performance can vary significantly across applications .

• Sample preparation optimization:

  • For Western blot: Test different lysis buffers (RIPA, NP-40, urea-based)

  • For IHC: Compare different fixatives and antigen retrieval methods

  • For IF: Test various permeabilization and fixation protocols

• Epitope accessibility issues: Consider that the TBRG1 epitope might be differentially accessible depending on sample preparation method. Test antibodies targeting different regions of TBRG1.

• Batch-to-batch variation: Request information on lot-specific validation data from manufacturers and maintain records of antibody performance by lot number.

• Cross-validation approach: Implement orthogonal validation methods to confirm results obtained with antibody-based detection .

• Experimental conditions standardization: Develop detailed standard operating procedures for each application to minimize technical variability.

Implementing these strategies can help ensure consistent and reliable results when working with TBRG1 antibodies across different experimental platforms.

How can researchers effectively use TBRG1 antibodies to explore its newly discovered role in invertebrate immunity?

The recent discovery of TBRG1's role in invertebrate immunity opens new research avenues . To effectively explore this:

• Cross-species validation: Validate existing TBRG1 antibodies for cross-reactivity with invertebrate homologs through Western blot analysis with recombinant proteins.

• Immune challenge experimental design:

  • Track TBRG1 expression changes following bacterial challenges using quantitative Western blot

  • Perform immunolocalization during immune responses to determine subcellular distribution changes

  • Correlate with antimicrobial peptide (AMP) expression through dual immunostaining

• Functional studies integration: Combine RNAi-mediated knockdown with antibody detection to confirm target reduction and correlate with bacteria clearance capacity.

• Evolutionary conservation analysis: Use validated antibodies to compare TBRG1 expression and localization patterns across evolutionary distant species during immune challenges.

• Pathway reconstruction: Employ co-immunoprecipitation with TBRG1 antibodies to identify species-specific interaction partners in immune pathways.

These approaches can help establish mechanistic connections between TBRG1 and innate immunity across evolutionary diverse organisms, potentially revealing conserved immune regulation mechanisms .

What methodological approaches are recommended for studying TBRG1 in the context of chromatin remodeling and transcriptional regulation?

Given the critical role of Brg1 (a distinct chromatin remodeling protein) in transcriptional regulation and development , researchers interested in potential similar functions of TBRG1 should consider:

• Chromatin immunoprecipitation (ChIP) optimization:

  • Develop optimized ChIP protocols specific for TBRG1 using validated antibodies

  • Implement dual crosslinking strategies (formaldehyde plus EGS) to capture indirect DNA associations

  • Validate ChIP efficiency using known target genes or regions

• Sequential ChIP (ChIP-reChIP): Employ sequential ChIP to identify co-occupancy with known transcriptional regulators or chromatin remodeling factors.

• Genome-wide binding studies: Combine ChIP with next-generation sequencing (ChIP-seq) to map TBRG1 binding sites across the genome in different cellular contexts.

• Transcriptional activity correlation: Correlate TBRG1 binding with transcriptional output using reporter assays or RNA-seq following TBRG1 modulation.

• Chromatin accessibility analysis: Pair TBRG1 ChIP-seq with ATAC-seq or DNase-seq to determine if TBRG1 binding correlates with changes in chromatin accessibility.

• Histone modification correlation: Analyze correlations between TBRG1 binding and specific histone modifications using sequential ChIP or parallel ChIP-seq experiments.

These approaches can help characterize potential roles of TBRG1 in chromatin dynamics and transcriptional regulation, possibly revealing functional similarities or differences with known chromatin remodelers like Brg1 .

How might TBRG1 antibodies be adapted for studying its potential role in infection and immunity against pathogens such as Mycobacterium tuberculosis?

Recent studies on antibody Fc features in Mycobacterium tuberculosis restriction suggest potential new avenues for studying TBRG1 in infection contexts:

• Infection model experimental design:

  • Track TBRG1 expression changes during bacterial infection using quantitative immunoblotting

  • Develop co-localization studies with pathogen markers using confocal microscopy

  • Analyze TBRG1 distribution in infected vs. uninfected cells using immunofluorescence

• Single-cell analysis approaches:

  • Adapt TBRG1 antibodies for flow cytometry or mass cytometry (CyTOF) applications

  • Implement intracellular staining protocols optimized for infected cells

  • Correlate TBRG1 expression with infection status at single-cell resolution

• Tissue-level analysis in infection models:

  • Optimize IHC protocols for detecting TBRG1 in infected tissues

  • Implement multiplex immunostaining to correlate with immune cell markers

  • Quantify spatial relationships between TBRG1-expressing cells and infection foci

• Functional correlation studies:

  • Design RNAi or CRISPR-based TBRG1 modulation in infection models

  • Use validated antibodies to confirm knockdown/knockout efficiency

  • Correlate TBRG1 levels with bacterial restriction capacity