TMSB4X Antibody, FITC conjugated

What is TMSB4X and why is it relevant for research?

TMSB4X (Thymosin beta-4) is a protein involved in multiple biological processes, most notably as a regulator of inflammation-associated ferroptosis. Recent studies have identified TMSB4X as a potential biomarker and therapeutic target for hepatocellular carcinoma (HCC), where it appears to promote cell viability, migration, and invasion while repressing ferroptosis in HCC cells . The protein has become increasingly important in cancer research, particularly in understanding the complex interplay between inflammation and ferroptosis in tumor progression.

What are the key specifications of commercial TMSB4X antibodies with FITC conjugation?

The TMSB4X antibody with FITC conjugation (e.g., ABIN7172133) is typically a polyclonal antibody raised in rabbits against the amino acid sequence 6-44 of human Thymosin beta-4. These antibodies are highly purified (>95% using Protein G purification) and formulated in liquid buffer containing preservatives such as 0.03% Proclin 300 and 50% Glycerol in 0.01M PBS at pH 7.4 . The FITC conjugation enables direct fluorescent detection, making it valuable for immunofluorescence and flow cytometry applications.

What is the epitope specificity of TMSB4X antibodies and why is it important?

Commercial TMSB4X antibodies target different epitopes within the protein, with specific antibodies recognizing amino acids 6-44, 1-44, 1-43, 38-43, or 1-11 . This epitope specificity is crucial as different functional domains of TMSB4X may be involved in different biological processes. When designing experiments, researchers should select antibodies with epitope specificity relevant to the protein domain of interest, particularly when studying protein-protein interactions or specific post-translational modifications.

What are the validated applications for TMSB4X antibodies?

TMSB4X antibodies have been validated for multiple applications including Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF), Immunoprecipitation (IP), and Enzyme-Linked Immunosorbent Assay (ELISA) . The specific applications vary by antibody clone and conjugation. For FITC-conjugated antibodies, the primary applications are flow cytometry and direct immunofluorescence microscopy, though researchers should validate the antibody for their specific experimental conditions.

How should TMSB4X antibodies be stored and handled to maintain efficacy?

TMSB4X antibodies, including FITC-conjugated versions, should typically be stored at -20°C for long-term storage and at 4°C for short-term use. It's important to avoid repeated freeze-thaw cycles, which can degrade antibody quality. When working with FITC-conjugated antibodies, researchers should protect them from light to prevent photobleaching of the fluorophore. Aliquoting the antibody upon first use is recommended to minimize freeze-thaw cycles and maintain antibody integrity .

What species reactivity can be expected with TMSB4X antibodies?

Different TMSB4X antibodies show varied species reactivity. Some antibodies react only with human TMSB4X, while others demonstrate cross-reactivity with mouse and rat homologs . When planning experiments with animal models, researchers should carefully select antibodies with validated reactivity for the species of interest. It's advisable to perform pilot experiments to confirm reactivity if working with species not explicitly validated by the manufacturer.

How can TMSB4X antibodies be utilized to study ferroptosis mechanisms?

To investigate TMSB4X's role in ferroptosis, researchers can employ FITC-conjugated TMSB4X antibodies in combination with ferroptosis inducers (e.g., erastin, RSL3) and inhibitors (e.g., ferrostatin-1). Flow cytometry using these antibodies can measure TMSB4X expression levels in cells undergoing ferroptosis. Co-localization studies using confocal microscopy can reveal interactions between TMSB4X and other ferroptosis-related proteins. Researchers should consider using siRNA knockdown of TMSB4X (e.g., with sequences like 5′-TAGCTGTTTAACTTTGTAAGATG-3′) to assess functional consequences on ferroptotic cell death markers .

What methodological approaches are optimal for investigating TMSB4X in inflammation-associated cancer progression?

For studying TMSB4X in inflammation-associated cancer progression, a multi-omic approach is recommended. Researchers should combine immunofluorescence using FITC-conjugated TMSB4X antibodies with transcriptomic analysis of inflammation markers. Tissue microarrays can be employed for examining TMSB4X expression patterns across cancer stages. The relationship between TMSB4X and inflammatory signaling can be assessed through phospho-protein arrays before and after TMSB4X modulation. Additionally, co-culture systems of cancer cells with immune cells can reveal how TMSB4X influences inflammatory microenvironments, with the FITC conjugation allowing direct visualization of protein localization during cell-cell interactions .

How can TMSB4X antibodies contribute to biomarker development for hepatocellular carcinoma?

To develop TMSB4X as a biomarker for HCC, researchers should design sequential studies starting with tissue microarray analysis using immunohistochemistry to correlate TMSB4X expression with clinical outcomes. FITC-conjugated TMSB4X antibodies can be employed in flow cytometry to quantify expression in circulating tumor cells or exosomes from patient blood samples. Multiplex immunofluorescence panels incorporating TMSB4X-FITC alongside other HCC markers can improve diagnostic accuracy. Advanced machine learning algorithms similar to those used in recent studies can be applied to integrate TMSB4X expression data with other clinical parameters to create prognostic models for HCC patients .

What controls should be included when using FITC-conjugated TMSB4X antibodies?

When designing experiments with FITC-conjugated TMSB4X antibodies, researchers should include:

• Isotype controls (FITC-conjugated rabbit IgG at matching concentrations) to assess non-specific binding

• Blocking controls using recombinant TMSB4X protein to confirm specificity

• Negative controls using cells/tissues known not to express TMSB4X

• Positive controls using cells/tissues with validated TMSB4X expression

• Additional controls when performing knockdown/overexpression studies to verify antibody specificity

• Spectral controls to account for autofluorescence, particularly in liver tissues which often exhibit high background fluorescence

What are the optimal fixation and permeabilization protocols for TMSB4X immunofluorescence studies?

For optimal immunofluorescence studies using FITC-conjugated TMSB4X antibodies, researchers should:

• Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

• Perform antigen retrieval if necessary, especially for tissue sections

• Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes (the exact concentration should be optimized)

• Block with 5% normal serum from the same species as the secondary antibody

• Incubate with TMSB4X-FITC antibody at optimized concentration (typically 1:100 to 1:500) overnight at 4°C

• Wash thoroughly to remove unbound antibody

• Mount with anti-fade mounting medium containing DAPI for nuclear counterstaining

• Store slides protected from light at 4°C for short-term or -20°C for long-term preservation

How should antibody concentration be optimized for different experimental platforms?

Optimizing antibody concentration requires systematic titration specific to each application:

Researchers should perform preliminary experiments with dilution series, analyze signal-to-noise ratios, and select the concentration that provides optimal specific signal with minimal background .

How can researchers address high background issues when using FITC-conjugated TMSB4X antibodies?

High background is a common challenge when using FITC-conjugated antibodies. To address this issue:

• Increase blocking time (2-3 hours) and concentration (up to 10% normal serum)

• Add 0.1-0.3% Triton X-100 to blocking and antibody dilution buffers

• Include 0.1-0.5% BSA in washing buffers to reduce non-specific binding

• Optimize antibody concentration - excessive antibody often increases background

• For tissues with high autofluorescence (especially liver), consider using Sudan Black B (0.1-0.3%) for 10 minutes after antibody incubation

• If cross-reactivity is suspected, pre-absorb the antibody with cell/tissue lysates from species showing cross-reactivity

• Consider using alternative fluorophores with different excitation/emission profiles if tissue autofluorescence overlaps with FITC spectrum

What approaches should be used to quantify TMSB4X expression in immunofluorescence studies?

For rigorous quantification of TMSB4X expression:

• Capture images with consistent exposure settings across all samples

• Use appropriate software (ImageJ, CellProfiler, or dedicated microscopy software)

• Define regions of interest (ROIs) based on biological relevance (e.g., cell membrane, cytoplasm, nucleus)

• Measure mean fluorescence intensity (MFI) within ROIs

• Subtract background fluorescence from regions without cells

• Normalize to cell number or area

• For co-localization studies, calculate Pearson's or Mander's correlation coefficients

• Perform statistical analysis using appropriate tests (t-test, ANOVA) with correction for multiple comparisons

• Present data with clear indication of sample size, biological replicates, and statistical significance

How can researchers validate contradictory results between different TMSB4X detection methods?

When facing contradictory results:

• Verify antibody specificity using alternative approaches:

  • Test multiple antibodies targeting different epitopes of TMSB4X

  • Perform siRNA knockdown experiments to confirm signal reduction

  • Use recombinant TMSB4X protein as a blocking control

• Employ orthogonal techniques:

  • Compare protein expression (antibody-based) with mRNA expression (qPCR)

  • Validate key findings with mass spectrometry

• Consider biological variables:

  • Assess protein localization changes under different conditions

  • Examine post-translational modifications that might affect antibody binding

  • Test for the presence of TMSB4X isoforms or splice variants

• Review methodological details:

  • Evaluate fixation and permeabilization effects on epitope accessibility

  • Consider buffer composition and pH effects on antibody binding

  • Assess potential effects of sample preparation on protein expression or structure

What is the current understanding of TMSB4X's role in ferroptosis regulation?

Recent research has identified TMSB4X as a regulator of inflammation-associated ferroptosis, particularly in hepatocellular carcinoma. Studies using bioinformatics analysis and machine learning algorithms have shown that TMSB4X is highly expressed in HCC samples compared to normal tissues. Functional studies demonstrate that TMSB4X promotes cancer cell viability, migration, and invasion while repressing ferroptosis in HCC cells. These findings suggest that TMSB4X may protect cancer cells from ferroptotic cell death, potentially contributing to tumor progression. The specific molecular mechanisms through which TMSB4X regulates ferroptosis remain under investigation, but current evidence suggests it may influence lipid peroxidation pathways or modulate iron metabolism .

How does TMSB4X interact with inflammatory signaling pathways in cancer?

The interaction between TMSB4X and inflammatory signaling in cancer appears multifaceted. Research using weighted gene co-expression network analysis (WGCNA) has identified TMSB4X among 157 genes related to both inflammation and ferroptosis in HCC. This suggests TMSB4X functions at the intersection of these two processes. Lipid peroxides generated during ferroptosis can serve as signals to activate dendritic cells and cytotoxic T cells, indicating a potential role in tumor immunotherapy. Conversely, inflammation can contribute to ferroptosis activation. While specific inflammatory pathways directly regulated by TMSB4X are still being elucidated, current evidence suggests it may modulate NF-κB signaling or cytokine production. Further research is needed to fully characterize the molecular interactions between TMSB4X and specific inflammatory mediators .

What prognostic value does TMSB4X expression have in cancer patients?

Machine learning algorithms, particularly the rLasso algorithm, have been used to develop prognostic models for HCC patients based on inflammation-associated ferroptosis regulators, with TMSB4X identified as the most important gene dominating the classification. The risk model constructed using TMSB4X and other related genes has shown good efficacy in predicting the clinical outcomes of HCC patients. Patients classified into high-risk and low-risk groups based on this model demonstrate distinct molecular characteristics. These findings suggest that TMSB4X expression levels could serve as a valuable prognostic biomarker for HCC and potentially other cancer types. Further validation in larger, diverse patient cohorts is needed to confirm the clinical utility of TMSB4X as a prognostic indicator .

What emerging therapeutic strategies target TMSB4X in cancer treatment?

Emerging therapeutic approaches targeting TMSB4X include:

• RNA interference strategies using specific siRNAs (such as 5′-TAGCTGTTTAACTTTGTAAGATG-3′ and 5′-CCCCTTTCACATCAAAGAACTAC-3′) to knockdown TMSB4X expression

• Combination therapies leveraging TMSB4X inhibition with ferroptosis inducers to enhance cancer cell death

• Development of small molecule inhibitors targeting TMSB4X-protein interactions

• Immunotherapeutic approaches exploiting the relationship between TMSB4X, inflammation, and tumor immunity