TMSB4X Antibody, HRP conjugated

What is TMSB4X protein and why is it important in research?

TMSB4X (thymosin beta 4 X-linked) is a small protein with 44 amino acid residues and a molecular weight of approximately 5.1 kDa. It is primarily localized in the cytoplasm and belongs to the Thymosin beta protein family. The protein plays a crucial role in cytoskeletal organization by binding to G-actin and preventing its polymerization, thereby regulating actin dynamics essential for cell motility, division, and differentiation. TMSB4X is expressed in several hemopoietic cell lines and lymphoid malignant cells, making it an important target in cancer research, particularly studies involving cell migration and invasion .

What are the key experimental applications of HRP-conjugated TMSB4X antibodies?

HRP-conjugated TMSB4X antibodies are primarily utilized in enzyme-linked detection methods where the horseradish peroxidase enzyme catalyzes colorimetric, chemiluminescent, or fluorescent reactions. The main applications include:

• Western Blot (WB): Detection of TMSB4X protein in cell or tissue lysates with enhanced sensitivity due to signal amplification by HRP

• Enzyme-Linked Immunosorbent Assay (ELISA): Quantification of TMSB4X levels in biological samples

• Immunohistochemistry (IHC): Visualization of TMSB4X protein localization in tissue sections

• Immunocytochemistry (ICC): Detection of TMSB4X in cultured cells

What cross-reactivity should researchers consider when using TMSB4X antibodies?

Researchers should be aware that TMSB4X antibodies may exhibit cross-reactivity across different species. Based on available commercial antibodies, many TMSB4X antibodies demonstrate reactivity with human, mouse, and rat TMSB4X proteins due to the high conservation of the protein sequence across species. Some antibodies also show cross-reactivity with bovine, horse, and other mammalian species. When designing experiments, verification of species-specific reactivity is essential, especially when working with non-human models. Additionally, researchers should validate the absence of cross-reactivity with other thymosin family proteins, particularly thymosin beta 10, which shares structural similarities with TMSB4X .

What are the optimal conditions for Western blot analysis using HRP-conjugated TMSB4X antibodies?

For optimal Western blot detection of TMSB4X using HRP-conjugated antibodies, researchers should follow these methodological guidelines:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell/tissue lysis

  • For this small protein (5.1 kDa), include preparation steps to prevent protein loss

  • Load 20-40 μg of total protein per well

• Gel electrophoresis:

  • Use 15-20% SDS-PAGE gels or specialized Tricine-SDS gels designed for small proteins

  • Include a reducing agent (β-mercaptoethanol or DTT) in sample buffer

• Transfer conditions:

  • Perform semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight

  • Use 0.2 μm PVDF membrane (preferred over nitrocellulose for small proteins)

• Antibody incubation:

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

  • Dilute primary antibody according to manufacturer's recommendation (typically 1:500-1:2000)

  • Incubate with HRP-conjugated secondary antibody if using unconjugated primary

  • For direct HRP-conjugated TMSB4X antibodies, a single incubation step is sufficient

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Adjust exposure time (typically 30 seconds to 5 minutes) based on signal intensity

How can TMSB4X antibodies be effectively used in immunohistochemistry applications?

To achieve optimal immunohistochemical detection of TMSB4X in tissue sections:

• Tissue preparation:

  • Fix tissues in 10% neutral-buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Cut sections at 4-5 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker method: 3 minutes at high pressure or microwave method: 20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5% normal serum

  • For HRP-conjugated antibodies, apply at manufacturer-recommended dilution (typically 1:50-1:200)

  • Incubate at 4°C overnight or at room temperature for 1-2 hours

• Detection and visualization:

  • For HRP-conjugated antibodies, apply DAB or AEC substrate directly

  • Counterstain with hematoxylin for nuclear visualization

  • Mount with appropriate mounting medium

• Controls:

  • Include positive control tissues (lymphoid tissues or hemopoietic cell lines)

  • Include negative controls by omitting primary antibody

  • Consider using tissues from TMSB4X knockout models as definitive negative controls

What quantitative analysis methods are recommended for ELISA-based detection of TMSB4X?

For quantitative analysis of TMSB4X using ELISA methodology:

• Assay setup:

  • Sandwich ELISA: Coat plates with capture antibody (1-10 μg/ml in carbonate buffer pH 9.6)

  • Direct ELISA: Coat plates with sample diluted in coating buffer

  • Block with 1-5% BSA or suitable blocking reagent

• Detection system:

  • For HRP-conjugated TMSB4X antibodies: Apply directly as detection antibody

  • Develop with TMB substrate and stop with 2N H₂SO₄

  • Read absorbance at 450 nm with 570 nm reference wavelength

• Standard curve preparation:

  • Use recombinant TMSB4X protein at concentrations ranging from 0.1-1000 ng/ml

  • Prepare 7-8 point standard curve with 2-fold or 3-fold serial dilutions

  • Include blank controls

• Data analysis:

  • Generate 4-parameter logistic curve fit for standard curve

  • Calculate sample concentrations using regression equation

  • Apply appropriate dilution factors

  • Perform spike-recovery tests to verify assay accuracy

• Sensitivity and range:

  • Typical detection range: 0.5-500 ng/ml

  • Lower limit of detection: approximately 0.1-0.5 ng/ml depending on antibody quality

How can researchers optimize dual immunofluorescence staining with TMSB4X antibodies to study colocalization with cytoskeletal elements?

For optimal dual immunofluorescence staining to study TMSB4X colocalization with cytoskeletal proteins:

• Sample preparation:

  • For cultured cells: Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

  • For tissue sections: Use freshly frozen sections or paraffin sections with appropriate antigen retrieval

• Blocking and primary antibody application:

  • Block with 5% normal serum from species unrelated to both primary antibodies

  • Apply unconjugated TMSB4X antibody together with anti-cytoskeletal protein antibody (e.g., anti-actin, anti-tubulin)

  • Incubate overnight at 4°C in humidified chamber

• Secondary antibody selection:

  • Select fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • Recommended combinations: TMSB4X (green/Alexa 488) + cytoskeletal marker (red/Alexa 594)

  • Include proper controls for antibody cross-reactivity

• Imaging parameters:

  • Use confocal microscopy with sequential scanning to prevent bleed-through

  • Optimize pinhole settings (0.7-1.0 Airy units)

  • Capture Z-stacks with 0.3-0.5 μm steps for 3D colocalization analysis

• Colocalization analysis:

  • Calculate Pearson's correlation coefficient and Mander's overlap coefficient

  • Use object-based colocalization for more accurate quantification

  • Apply appropriate thresholding methods consistently across samples

What methodological approaches can resolve contradictory findings regarding TMSB4X expression in different cancer types?

To address contradictory findings regarding TMSB4X expression across cancer types:

• Multi-omics validation strategy:

  • Compare protein expression (Western blot, IHC, ELISA) with mRNA expression (qRT-PCR, RNA-seq)

  • Validate with multiple antibodies targeting different epitopes of TMSB4X

  • Use mass spectrometry-based proteomics as an antibody-independent method

• Context-specific analysis:

  • Compare TMSB4X expression across different cellular compartments (cytoplasmic vs. nuclear)

  • Assess expression in tumor cells vs. stromal/immune cells using dual staining

  • Evaluate expression at different stages of cancer progression

• Methodological standardization:

  • Standardize tissue processing protocols across studies

  • Use consistent scoring systems for IHC evaluation

  • Apply digital pathology and automated image analysis for objective quantification

• Functional validation:

  • Perform gene knockdown and overexpression studies in multiple cell lines

  • Evaluate phenotypic changes in proliferation, migration, and invasion

  • Correlate expression with clinical outcomes in well-characterized patient cohorts

• Technical considerations for hepatocellular carcinoma (HCC) studies:

  • Address HCC tissue heterogeneity by sampling multiple tumor regions

  • Include adjacent non-tumor tissue controls from the same patients

  • Consider cirrhosis status as a confounding variable in expression analysis

How can researchers investigate the role of TMSB4X in inflammation-associated ferroptosis using HRP-conjugated antibodies?

To investigate TMSB4X's role in inflammation-associated ferroptosis:

• Experimental design for ferroptosis detection:

  • Induce ferroptosis using erastin, RSL3, or sorafenib in HCC cell lines

  • Measure lipid peroxidation using C11-BODIPY or MDA assays

  • Assess cell viability using standard assays (MTT, CCK-8)

  • Measure GSH levels and GPX4 activity as ferroptosis markers

• TMSB4X manipulation:

  • Perform knockdown using siRNA or CRISPR-Cas9

  • Overexpress TMSB4X using appropriate expression vectors

  • Create stable cell lines with inducible TMSB4X expression

• Protein interaction studies:

  • Use HRP-conjugated TMSB4X antibodies for co-immunoprecipitation followed by Western blot

  • Perform proximity ligation assay to detect TMSB4X interaction with ferroptosis regulators

  • Validate interactions using recombinant proteins in vitro

• Inflammatory signaling analysis:

  • Measure inflammatory cytokine levels (IL-1β, IL-6, TNF-α) in culture supernatants

  • Analyze NF-κB pathway activation using phospho-specific antibodies

  • Assess NLRP3 inflammasome activation in relation to TMSB4X expression

• In vivo validation:

  • Develop xenograft models with TMSB4X-modified HCC cells

  • Perform IHC for TMSB4X and ferroptosis markers

  • Correlate TMSB4X expression with inflammatory markers and tumor progression

What are the most common issues when using HRP-conjugated TMSB4X antibodies, and how can researchers resolve them?

Common issues and their solutions when working with HRP-conjugated TMSB4X antibodies:

• Low signal intensity:

  • Increase antibody concentration incrementally (1.5-2x manufacturer recommendation)

  • Extend incubation time to overnight at 4°C

  • Use signal enhancement systems (biotinyl tyramide amplification)

  • Ensure appropriate antigen retrieval for fixed tissues

  • Check HRP activity with direct enzyme assay

• High background:

  • Increase blocking time and concentration (use 5% BSA instead of 1-3%)

  • Add 0.1-0.3% Triton X-100 to washing buffer

  • Pre-absorb antibody with tissue powder

  • Reduce primary antibody concentration

  • Include 0.05-0.1% Tween-20 in antibody diluent

• Non-specific bands in Western blot:

  • Perform peptide competition assay to identify specific bands

  • Use gradient gels to better resolve proteins of similar molecular weight

  • Optimize transfer conditions for small proteins

  • Increase washing time and buffer volume

• Inconsistent results across experiments:

  • Prepare master mixes of reagents for technical replicates

  • Standardize protein extraction methods

  • Use internal loading controls consistently

  • Control for lot-to-lot variations in antibodies

  • Implement positive and negative controls in each experiment

How should researchers validate TMSB4X antibody specificity for their experimental systems?

Comprehensive validation strategies for TMSB4X antibody specificity:

• Genetic validation:

  • Test antibody in TMSB4X knockout or knockdown models

  • Use overexpression systems as positive controls

  • Compare staining patterns in cells with known differential expression

• Peptide competition assays:

  • Pre-incubate antibody with excess TMSB4X peptide (10-100x molar excess)

  • Compare results with and without competing peptide

  • Use unrelated peptide as negative control

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes

  • Include monoclonal and polyclonal antibodies in validation

  • Cross-validate with different detection methods

• Orthogonal method validation:

  • Compare protein detection with mRNA expression (qPCR or in situ hybridization)

  • Correlate with mass spectrometry-based protein identification

  • Use recombinant TMSB4X protein as standard

• Species-specific validation:

  • Verify cross-reactivity with target species using sequence alignment

  • Test reactivity in multiple cell lines from the species of interest

  • Create dilution series with recombinant proteins from different species

What considerations are important when selecting between direct HRP-conjugated antibodies versus two-step detection methods?

Selection considerations for direct HRP-conjugated TMSB4X antibodies versus two-step detection:

• Sensitivity requirements:

  Detection Method	Sensitivity	Signal-to-Noise	Detection Limit
  Direct HRP-conjugated	Moderate	Moderate	~1-5 ng protein
  Two-step (primary + secondary)	Higher	Higher	~0.1-1 ng protein
  Three-step (biotin-streptavidin)	Highest	Variable	~0.01-0.1 ng protein

• Application-specific considerations:

  • Western blot: Two-step methods generally provide better sensitivity for low abundance proteins

  • IHC: Direct methods reduce background from endogenous biotin and non-specific binding

  • ELISA: Direct methods simplify workflow but may reduce detection range

  • Multiplexing: Direct methods allow simpler multiplex design with multiple conjugated antibodies

• Experimental constraints:

  • Time considerations: Direct methods reduce protocol time by ~2 hours

  • Sample limitations: Two-step methods consume less primary antibody

  • Cross-reactivity concerns: Direct methods eliminate secondary antibody cross-reactivity

  • Signal amplification needs: Two-step methods allow for signal enhancement

• Technical factors:

  • Conjugation impact on epitope binding

  • Stability of HRP-conjugated antibodies (typically shorter shelf-life)

  • Lot-to-lot variation in conjugation efficiency

  • Availability of optimized conjugates for specific applications

What is the current understanding of TMSB4X's role in hepatocellular carcinoma progression based on recent studies?

Recent research has established TMSB4X as a significant regulator in hepatocellular carcinoma (HCC) progression:

How can researchers design experiments to elucidate the molecular mechanisms connecting TMSB4X to ferroptosis regulation?

Experimental design to investigate TMSB4X-ferroptosis connections:

• Ferroptosis induction and assessment:

  • Treat HCC cell lines with ferroptosis inducers (erastin, RSL3) at varying concentrations (0.1-10 μM)

  • Modify TMSB4X expression through overexpression and knockdown

  • Measure cell death via flow cytometry (Annexin V/PI staining)

  • Assess lipid peroxidation (C11-BODIPY, TBARS assay)

  • Measure cellular iron content and ROS levels

• Molecular pathway analysis:

  • Western blot analysis of key ferroptosis pathway components:

    • GPX4 expression and activity

    • System Xc- components (SLC7A11)

    • Iron metabolism regulators (FTH1, TFRC)

    • Lipid metabolism enzymes (ACSL4, LPCAT3)

  • qPCR analysis of ferroptosis-related gene expression

  • Lipidomic analysis to identify changes in membrane phospholipid composition

• Protein interaction studies:

  • Co-immunoprecipitation to identify TMSB4X binding partners in ferroptosis pathway

  • Proximity ligation assay to visualize protein interactions in situ

  • CRISPR-Cas9 screening to identify genetic dependencies

• Transcriptomic and proteomic profiling:

  • RNA-seq and proteomics analysis of TMSB4X-modulated cells

  • Pathway enrichment analysis focusing on redox regulation

  • Integration with existing ferroptosis gene signatures

• In vivo models:

  • Generate xenograft models with TMSB4X-modulated HCC cells

  • Treat with ferroptosis inducers and measure tumor response

  • Analyze tumor tissues for markers of ferroptosis and inflammation

What implications do recent discoveries about TMSB4X have for developing novel therapeutic approaches for cancer?

Therapeutic implications of TMSB4X research findings:

• TMSB4X as a therapeutic target:

  • Potential strategies for TMSB4X inhibition:

    • Small molecule inhibitors disrupting TMSB4X-actin interaction

    • Peptide-based competitive inhibitors

    • RNA interference approaches (siRNA, antisense oligonucleotides)

  • Combination approaches with existing therapies:

    • Ferroptosis inducers (erastin, sorafenib)

    • Conventional chemotherapeutics

    • Immune checkpoint inhibitors

• Biomarker applications:

  • Prognostic stratification of HCC patients based on TMSB4X expression

  • Prediction of response to ferroptosis-inducing therapies

  • Monitoring treatment response through liquid biopsy approaches

• Drug discovery considerations:

  • Screening assays for compounds that modulate TMSB4X-mediated ferroptosis resistance

  • Development of ferroptosis sensitizers targeting TMSB4X-dependent pathways

  • Repurposing of approved drugs that may influence TMSB4X activity

• Translational challenges:

  • Tissue-specific functions of TMSB4X in different cell types

  • Potential off-target effects due to TMSB4X roles in normal tissue homeostasis

  • Delivery methods for TMSB4X-targeting therapeutics

  • Resistance mechanisms that might emerge during TMSB4X-targeted therapy

• Future directions:

  • Development of TMSB4X activity bioassays for clinical samples

  • Integration of TMSB4X status in clinical trial design

  • Exploration of TMSB4X in cancer types beyond HCC

  • Investigation of TMSB4X in the tumor microenvironment and immune modulation

What experimental approaches would best elucidate the role of post-translational modifications of TMSB4X in its biological functions?

Experimental approaches to study TMSB4X post-translational modifications:

• Identification of modifications:

  • Mass spectrometry-based proteomic analysis:

    • Enrichment of TMSB4X from cell lysates via immunoprecipitation

    • Tryptic digestion and LC-MS/MS analysis

    • Targeted analysis for specific modifications (phosphorylation, acetylation, etc.)

  • Western blotting with modification-specific antibodies

  • 2D gel electrophoresis to separate protein isoforms

• Site-directed mutagenesis studies:

  • Generate mutants at predicted modification sites

  • Create phosphomimetic mutants (Ser/Thr to Asp/Glu)

  • Develop non-modifiable mutants (Ser/Thr to Ala)

  • Assess functional consequences in cellular assays

• Temporal dynamics of modifications:

  • Pulse-chase experiments with modification-specific detection

  • Time-course analysis following stimulation

  • Single-cell analysis using phospho-specific antibodies

  • Development of FRET-based biosensors for real-time monitoring

• Enzyme identification:

  • Inhibitor screens to identify responsible kinases/phosphatases

  • Enzyme-substrate validation assays

  • Co-immunoprecipitation studies to detect enzyme-TMSB4X complexes

  • In vitro kinase/phosphatase assays with recombinant proteins

• Functional consequences:

  • Binding assays with modified vs. unmodified TMSB4X

  • Structural studies (CD spectroscopy, NMR) to assess conformational changes

  • Cellular localization studies of modified TMSB4X

  • Differential interactome analysis using BioID or proximity labeling approaches

How might single-cell analysis technologies advance our understanding of TMSB4X expression patterns in heterogeneous tumor microenvironments?

Applications of single-cell technologies for TMSB4X research:

• Single-cell RNA sequencing (scRNA-seq):

  • Characterize cell-type specific expression patterns of TMSB4X in tumor tissues

  • Identify rare cell populations with distinct TMSB4X expression

  • Construct pseudo-time trajectories to understand TMSB4X expression during cellular differentiation

  • Integration with spatial transcriptomics data

• Single-cell proteomics approaches:

  • Mass cytometry (CyTOF) with TMSB4X antibodies

  • Multiplexed ion beam imaging (MIBI) for spatial resolution

  • Single-cell Western blotting for protein isoform detection

  • Microfluidic-based single-cell secretomics

• Spatial transcriptomics and proteomics:

  • In situ hybridization techniques (RNAscope, MERFISH)

  • Digital spatial profiling

  • Spatial mapping of TMSB4X in relation to vascular structures and immune infiltrates

  • Multiplexed immunofluorescence with machine learning-based image analysis

• Functional single-cell assays:

  • Live cell imaging with TMSB4X reporters

  • Single-cell secretion assays for inflammation markers

  • Cell migration tracking in relation to TMSB4X expression

  • Single-cell ferroptosis sensitivity assays

• Computational integration:

  • Multi-omics data integration at single-cell resolution

  • Trajectory inference algorithms to map TMSB4X dynamics

  • Cell-cell communication analysis based on receptor-ligand interactions

  • Deconvolution of bulk tissue data using single-cell reference maps

What methodological advances are needed to better understand the interplay between TMSB4X, inflammation, and ferroptosis in different disease contexts?

Methodological advances needed for TMSB4X research in disease contexts:

• Improved detection systems:

  • Development of high-affinity, highly specific monoclonal antibodies

  • Creation of nanobody-based detection reagents for live imaging

  • Improved TMSB4X activity assays beyond simple binding measurements

  • Biosensors to detect TMSB4X-actin interaction dynamics in real-time

• Advanced disease models:

  • Patient-derived organoids with defined genetic backgrounds

  • Humanized mouse models for studying immune interactions

  • CRISPR-engineered models with endogenous TMSB4X tagging

  • Tissue-specific conditional knockout models

• Multi-parametric ferroptosis assessment:

  • Simultaneous measurement of multiple ferroptosis markers

  • Live-cell imaging methods for ferroptosis progression

  • Correlative light and electron microscopy for ultrastructural analysis

  • In vivo ferroptosis detection methods

• Inflammation monitoring:

  • Multiplexed cytokine profiling in relation to TMSB4X expression

  • Single-cell inflammasome activation assays

  • Live tracking of inflammatory cell recruitment

  • Non-invasive imaging of inflammation in animal models

• Translational methodologies:

  • Development of companion diagnostic assays for TMSB4X status

  • Liquid biopsy approaches for monitoring TMSB4X-related biomarkers

  • Standardized scoring systems for TMSB4X expression in clinical samples

  • AI-assisted image analysis for TMSB4X expression patterns in tumors