TALDO1 Antibody

What is TALDO1 and what is its biological significance in cellular metabolism?

TALDO1 (Transaldolase 1) is a key enzyme in the nonoxidative phase of the pentose phosphate pathway (PPP). It catalyzes the reversible conversion of sedoheptulose-7-phosphate and D-glyceraldehyde-3-phosphate into erythrose-4-phosphate and beta-D-fructose-6-phosphate, contributing significantly to the generation of NADPH . Beyond its metabolic role, TALDO1 influences various cellular pathways by altering its subcellular localization between the nucleus and cytoplasm . Deficiency in TALDO1 has been associated with liver cirrhosis, hepatosplenomegaly, and other clinical manifestations including telangiectases of the skin and enlarged clitoris .

What applications are TALDO1 antibodies typically validated for?

TALDO1 antibodies have been validated for multiple experimental applications:

It's recommended to titrate the antibody in each testing system to obtain optimal results as response may be sample-dependent .

What is the molecular structure and weight of TALDO1?

TALDO1 has a calculated molecular weight of 37.39 kDa, consisting of 337 amino acids . Interestingly, TALDO1 exists in two isoforms generated by alternative translational initiation. The long isoform (TALDO1L) contains all 337 amino acids, while the short isoform (TALDO1S) lacks the first 10 N-terminal amino acids . When performing Western blot analysis, these two isoforms appear as two distinct bands around 37 kDa, with varying expression levels and ratios among different cell lines .

How should researchers optimize antibody dilutions for different experimental applications?

For TALDO1 antibody applications, follow these methodological considerations:

• Western Blotting: Begin with a 1:2000 dilution and adjust based on signal intensity. For cell lines with known high TALDO1 expression (such as HepG2), consider using dilutions up to 1:4000. For tissues or cells with potentially lower expression, start with 1:1000 .

• Immunohistochemistry: For paraffin-embedded tissues, start with a 1:500 dilution. Antigen retrieval is critical - use TE buffer (pH 9.0) as the primary method, with citrate buffer (pH 6.0) as an alternative if results are suboptimal . Optimization may be needed for different tissue types.

• Immunoprecipitation: Use 2.0 μg of antibody per 2.0 mg of total protein lysate as a starting point, and adjust based on pull-down efficiency .

Regardless of application, always include positive controls (HepG2 or COLO 320 cells) and negative controls (TALDO1 knockout cells if available) to validate antibody specificity .

What are the recommended storage conditions to maintain TALDO1 antibody stability?

To ensure optimal performance of TALDO1 antibodies:

• Store at -20°C in buffer containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) .

• The antibody remains stable for one year after shipment when properly stored.

• Aliquoting is generally unnecessary for -20°C storage, although it may be advisable if the antibody will be used numerous times to avoid freeze-thaw cycles .

• Note that some preparations (20μl sizes) may contain 0.1% BSA, which should be considered when planning experiments .

How can researchers distinguish between the two TALDO1 isoforms (TALDO1L and TALDO1S) in experimental settings?

Differentiating between TALDO1 isoforms requires specific experimental strategies:

• Western Blot Analysis: Standard Western blot using anti-TALDO1 antibodies typically reveals two bands around 37 kDa. The upper band represents TALDO1L, while the lower band represents TALDO1S .

• Site-Directed Mutagenesis Approach: To confirm isoform identity, researchers can create single alanine substitution mutants:

  • M1A mutation (substitution at the first methionine codon) will diminish the upper band (TALDO1L) and enhance the lower band (TALDO1S)

  • M11A mutation (substitution at the second methionine codon) will abolish the lower band (TALDO1S)

  • S2A mutation (optimized first Kozak sequence) will enhance the upper band and nearly abolish the lower band

• Subcellular Localization Studies: The two isoforms show different subcellular distributions:

  • TALDO1L is distributed throughout the nucleus and cytoplasm

  • TALDO1S is predominantly localized in the cytoplasm when expressed alone, but may show altered distribution when co-expressed with TALDO1L due to heterodimerization

This approach not only confirms isoform identity but also provides insights into their functional differences.

What is the correlation between TALDO1 expression and hepatocellular carcinoma (HCC) progression?

TALDO1 has emerged as a potential biomarker in HCC, with significant clinical implications:

These findings collectively suggest that TALDO1 antibodies can be valuable tools in studying HCC progression and potentially in developing diagnostic or therapeutic strategies.

How can researchers effectively generate and validate TALDO1 knockout cell lines?

The generation of TALDO1 knockout cell lines provides valuable negative controls for antibody validation and enables functional studies. Based on published methodologies:

• CRISPR-Cas9 System Design:

  • Design target sequences using CRISPR Design Tools (e.g., http://crispr.mit.edu/)

  • A validated target sequence is 5'-ACCACCGTGGTGGCCGACAC-3' (positions 64-83 in the mouse TALDO1 gene)

  • Clone annealed oligonucleotides into appropriate vectors such as pSpCas9(BB)-2A-Puro

• Transfection and Selection:

  • Transfect target cells (e.g., NIH/3T3) using Lipofectamine® 2000

  • Apply puromycin selection (3 μg/mL) 24 hours post-transfection for approximately 3 days

  • Re-culture cells in fresh medium until colony formation

• Validation of Knockout Efficiency:

  • Genomic PCR against potential off-target genes (examples from published work include Wbscr16, Ikzf2, Thsd1, and Ttc28)

  • DNA sequencing of the TALDO1 gene region to confirm genomic alterations

  • Western blotting using anti-TALDO1 antibodies (absence of both TALDO1 bands confirms successful knockout)

  • Immunofluorescence staining

• Functional Validation:

  • Metabolic assays to confirm altered pentose phosphate pathway function

  • Incubation with ribose-5-phosphate followed by analysis of phosphate sugar metabolites can confirm TALDO1 deficiency

This comprehensive validation approach ensures the generation of reliable TALDO1 knockout cell lines for experimental use.

What is the relationship between TALDO1 and immune cell infiltration in the tumor microenvironment?

TALDO1 has been implicated in modulating the immune microenvironment, particularly in HCC:

• Relationship with CD4+ T Cells: TALDO1 expression shows a significant negative correlation with CD4+ T cell infiltration (Rho=-0.158, P=0.003) .

• Association with Regulatory T Cells: Conversely, TALDO1 demonstrates a significant positive correlation with regulatory T cell infiltration (Rho=0.134, P=0.012) .

• Immune Inhibitor/Stimulator Correlations: TALDO1 expression correlates with various immune markers:

These correlations suggest that TALDO1 may function as a negative immunoregulatory factor in HCC .

• Methodological Approaches for Study:

  • Utilize TIMER2 (http://timer.cistrome.org/) to explore relationships between TALDO1 expression and immune cell infiltration

  • Apply TISIDB (http://cis.hku.hk/TISIDB/) to investigate associations between TALDO1 and immune inhibitors/stimulators

  • Perform Spearman's rank correlation tests to calculate p-values and partial correlation coefficients

Understanding these relationships can inform immunotherapeutic approaches in TALDO1-expressing cancers.

How should researchers design experiments to investigate TALDO1's role in cancer cell proliferation and migration?

Based on published methodologies, the following experimental design is recommended:

• TALDO1 Knockdown:

  • Use siRNA-mediated gene silencing (si-TALDO1) with appropriate negative controls (si-NC)

  • Validate knockdown efficiency using both qPCR and Western blot

• Proliferation Assays:

  • CCK-8 Assay: Plate 2000 cells/well in 96-well plates. Add 10 μL CCK-8 solution and incubate for 2 hours at 37°C. Measure optical density at 450 nm at 0, 24, 48, and 72 hours .

  • EdU Assay: As a complementary method to confirm proliferation results .

• Migration Assays:

  • Transwell Assay: Assess migratory ability of control vs. TALDO1-knockdown cells .

• Mechanistic Investigation:

  • Examine EMT markers (e.g., E-cadherin) via Western blot

  • Assess MAPK pathway activation by measuring ERK1/2 and p-ERK1/2 levels

  • Use antibodies against TALDO1, E-cadherin, ERK1/2, p-ERK1/2, and appropriate loading controls (e.g., β-actin)

• Data Analysis:

  • Apply appropriate statistical tests

  • Consider correlating in vitro findings with clinical data from patient databases (TCGA, GEO)

This comprehensive approach allows for thorough investigation of TALDO1's functional role in cancer progression.

How can TALDO1 antibodies be utilized in the diagnosis of TALDO1 deficiency disorders?

TALDO1 deficiency is a rare inborn error of metabolism affecting the pentose phosphate pathway. Diagnostic approaches include:

• Clinical Presentation: Patients typically present with liver cirrhosis and hepatosplenomegaly during early infancy. Other features may include telangiectases of the skin and enlarged clitoris .

• Biochemical Markers: Elevated concentrations of ribitol, d-arabitol, and erythritol in urine and plasma are characteristic .

• Enzyme Activity Assay:

  • Isolate lymphoblasts or erythrocytes from patient samples

  • Incubate with ribose-5-phosphate

  • Analyze phosphate sugar metabolites to assess transaldolase activity

  • Reduced activity confirms TALDO1 deficiency

• Immunoblotting:

  • Use anti-TALDO1 antibodies to detect protein expression in patient samples

  • Compare with healthy controls to identify reduced or absent TALDO1 protein

• Genetic Analysis:

  • Sequence the TALDO1 gene to identify potential mutations

  • A reported pathogenic mutation is a homozygous 3 bp deletion resulting in the absence of serine at position 171, located in a conserved region critical for enzyme activity

This multimodal approach ensures accurate diagnosis of this rare metabolic disorder.

What methodological considerations should researchers follow when using TALDO1 as a prognostic biomarker in cancer research?

When utilizing TALDO1 as a potential prognostic biomarker, consider these methodological approaches:

• Gene Expression Analysis:

  • Analyze TALDO1 mRNA levels in tumor tissues compared to normal tissues using established databases (TCGA, GEO)

  • For HCC research, TCGA-LIHC data with 371 liver tissues and 50 non-tumor tissues, and GSE3637613 with 240 liver tissues and 193 normal tissues provide valuable resources

• Clinical Correlation:

  • Associate TALDO1 expression with clinicopathological features using appropriate statistical tests

  • Apply multivariate regression analysis to evaluate TALDO1 as an independent prognostic factor

  • Develop nomogram prognostic models incorporating TALDO1 expression with other clinical parameters

• Survival Analysis:

  • Perform Kaplan-Meier survival analysis comparing high vs. low TALDO1 expression groups

  • Use appropriate statistical methods (log-rank test, Cox regression) to determine significance

• Immunohistochemistry Protocol:

  • Use anti-TALDO1 antibodies at appropriate dilutions (1:200-1:800) on paraffin-embedded tissue sections

  • For optimal results with liver tissues, use antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0

  • Quantify staining intensity using established scoring systems

• Functional Validation:

  • Complement expression data with functional studies using cell lines

  • Apply TALDO1 knockdown or overexpression to evaluate effects on proliferation, migration, and relevant signaling pathways

This comprehensive approach provides robust evidence for TALDO1's potential as a prognostic biomarker.