TKTL2 Antibody

What is TKTL2 and what role does it play in cellular biology?

TKTL2 (Transketolase-like protein 2) is an enzyme that plays a critical role in cellular metabolism. Research indicates that TKTL2 is involved in total transketolase activity, which is essential for cancer cell proliferation. When transketolase activity is inhibited through mechanisms such as anti-TKTL1 siRNA transfection, total transketolase activity dramatically decreases and proliferation is significantly inhibited in cancer cells. TKTL2 has been identified as a pivotal factor in carcinogenesis and may contribute to tumor progression through both metabolic and non-metabolic pathways .

What types of TKTL2 antibodies are available for research and how do they differ?

Several types of TKTL2 antibodies are available for research purposes:

• Monoclonal antibodies: Such as Rabbit Recombinant Monoclonal TKTL2 antibody [EPR8592], which offers high specificity and reproducibility for human samples .

• Polyclonal antibodies: Including Rabbit Polyclonal antibodies targeting different epitopes:

  • Antibodies targeting amino acids 38-87 of human TKTL2

  • Antibodies targeting N-terminal regions (aa 43-71)

  • Antibodies purified by protein A chromatography methods

These antibodies differ in their specificity, host species, epitope targets, and recommended applications. The choice between monoclonal and polyclonal depends on research needs, with monoclonals offering greater specificity and reproducibility while polyclonals may provide broader epitope recognition .

How is TKTL2 expression regulated in normal versus cancer tissues?

Studies using The Cancer Genome Atlas (TCGA) datasets indicate that TKTL2 expression is significantly upregulated in several cancer types compared to normal tissues. In lung adenocarcinoma (LUAD), TKTL2 expression is significantly increased with a fold change of 0.025 (P<0.01). This upregulation pattern has been validated in paired tissue samples where TKTL2 mRNA levels were dramatically higher in cancer tissues than in adjacent normal tissues (P < 0.05). Immunohistochemistry studies have revealed that while TKTL1 shows no significant difference between normal and cancer tissues, TKTL2 tends to be elevated in carcinoma tissues. This differential expression suggests specific regulatory mechanisms that are altered during carcinogenesis .

What are the validated applications for TKTL2 antibodies in cancer research?

TKTL2 antibodies have been validated for several key applications in cancer research:

• Western Blotting (WB): Detection of TKTL2 protein expression in cell lysates including Jurkat, HepG2, BxPC-3, and MCF7 cancer cell lines at a recommended dilution of 1/1000. The predicted band size is approximately 68 kDa .

• Immunohistochemistry (IHC-P): Analysis of TKTL2 expression in paraffin-embedded tissues such as hepatocellular carcinoma at a dilution of 1/100. This technique requires heat-mediated antigen retrieval before staining .

• ELISA: Quantitative measurement of TKTL2 levels in various sample types, with reported titers as sensitive as 1:312500 using peptide-based assays .

• Prognostic Biomarker Research: TKTL2 antibodies are used to evaluate expression levels as potential prognostic markers in various cancers, including lung adenocarcinoma and ovarian cancer .

What is the optimal protocol for using TKTL2 antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) results with TKTL2 antibodies:

• Tissue Preparation:

  • Cut tissues into 4-μm-thick sections

  • Fix sections on slides and dry for 12-24 h at 37°C

  • Deparaffinize in xylene and rehydrate through graded ethanol and distilled water

• Antigen Retrieval:

  • Perform heat-mediated antigen retrieval (critical step for optimal results)

  • Use appropriate buffer (typically citrate or EDTA-based)

• Antibody Incubation:

  • Use anti-human TKTL2 antibody at a dilution of 1/100

  • Incubate overnight at 4°C

• Detection:

  • Incubate with appropriate secondary antibody

  • Add DAB chromogenic reagent

  • Mount slides after dehydration

• Evaluation:

  • Score intensity of immunostaining based on degree of color: none (0), yellow (1), brown and yellow (2), and tan (3)

  • Assess proportion of positive tumor cells: 0, 0%; 1, 1%-20%; 2, 21%-40%; 3, 41%-60%; 4, 61%-80%; and 5, 81%-100%

  • Calculate final score by multiplying intensity and percentage (range 0-15)

  • Classify as low (0-3) or high (4-15) expression

What are the best practices for Western blot analysis using TKTL2 antibodies?

For optimal Western blot results with TKTL2 antibodies:

• Sample Preparation:

  • Extract total cellular proteins with RIPA buffer

  • Load equal amounts of protein (30 μg/lane)

  • Separate by SDS-PAGE and transfer to PVDF membranes

• Blocking:

  • Block with PBS buffer containing 5% non-fat milk and 0.1% Tween 20

• Primary Antibody Incubation:

  • Dilute TKTL2 antibody at 1/1000 concentration in blocking buffer

  • Incubate overnight at 4°C

  • For some antibodies, recommended dilution is 1 μg/mL in 5% skim milk/PBS buffer

• Secondary Antibody:

  • Incubate with HRP-conjugated anti-Rabbit IgG (diluted 1:2000 to 1:50000)

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop with enhanced chemiluminescence

  • Expected band size for TKTL2 is approximately 68 kDa

• Validated Cell Lines:

  • Jurkat, HepG2, BxPC-3, and MCF7 cell lysates have been validated for TKTL2 detection

How can TKTL2 antibodies be used to investigate its role as a prognostic biomarker in cancer?

TKTL2 antibodies have proven valuable for investigating its potential as a prognostic biomarker through:

What mechanisms explain TKTL2's role in promoting cancer cell proliferation and migration?

Research into TKTL2 and related transketolase family members has revealed several mechanisms by which they promote cancer progression:

• Metabolic Reprogramming:

  • TKTL2 contributes to the Warburg effect in cancer cells

  • Supports nucleic acid synthesis through the pentose phosphate pathway

  • May interact with lactate dehydrogenase to support cancer metabolism

• Non-Metabolic Functions:

  • Nuclear localization of transketolase promotes proliferation, viability, and migration in a non-metabolic manner

  • Proteomic analyses (cross-linking Co-IP/MS) have revealed interactions with kinases and transcriptional coregulators

  • These include epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 3 (MAPK3)

  • These interactions are associated with cell activation and stress response processes

• Growth Factor Signaling:

  • EGF treatment significantly increases the viability of TKT wild-type cells

  • This effect can be blocked by EGFR inhibitor erlotinib treatment

  • TKTL2 may participate in these signaling pathways through its interactions with key kinases

How can inhibition of TKTL2 be studied using antibodies and inhibitors?

Studying TKTL2 inhibition can be approached through:

• Functional Antibody Studies:

  • Use antibodies to identify and validate TKTL2 expression before and after inhibition

  • Monitor changes in protein expression via Western blotting, IHC, or immunofluorescence

  • Correlate with functional assays to link expression to biological effects

• Small Molecule Inhibitors:

  • Thiamine analogs like oxythiamine (OT) can be used to inhibit transketolase activity

  • Growth curve analyses show that A549 cells are suppressed under different concentrations of inhibitors in a time-and dose-dependent manner

  • The percentage of apoptotic cells increases significantly with OT treatment

  • OT significantly induces G0/G1 arrest, increasing G0 to G1 phase cells (p<0.001) and decreasing S-phase population (p=0.004)

• Combined Therapeutic Approaches:

  • Antibodies can be used to monitor TKTL2 expression during combined treatment with conventional chemotherapeutics

  • Studies show that transketolase overexpression increases cisplatin resistance

  • Silencing or combined treatment with cisplatin could restore sensitivity

What are the key considerations for antibody storage and handling to maintain optimal activity?

For maintaining optimal TKTL2 antibody activity:

• Storage Conditions:

  • For lyophilized antibodies: Store at -20°C or below

  • For reconstituted antibodies: Aliquot and store at -20°C

  • Avoid multiple freeze/thaw cycles which can degrade antibody quality and performance

• Reconstitution Guidelines:

  • For lyophilized antibodies in PBS buffer with 2% sucrose: Add 100 μL of distilled water

  • Final antibody concentration after reconstitution is typically 1 mg/mL

  • Allow complete dissolution before use

• Handling Precautions:

  • Maintain sterile conditions when handling antibodies

  • Minimize exposure to light for conjugated antibodies

  • Follow manufacturer's guidelines for specific antibodies

  • Use appropriate buffers as recommended (typically PBS containing 0.09% sodium azide)

• Quality Control:

  • Periodically validate antibody performance using positive controls

  • Monitor for changes in specificity or sensitivity over time

How can researchers verify the specificity of TKTL2 antibodies and avoid cross-reactivity issues?

To ensure TKTL2 antibody specificity:

• Positive and Negative Controls:

  • Use validated cell lines with known TKTL2 expression (Jurkat, HepG2, BxPC-3, MCF7)

  • Include negative controls (tissues or cells without TKTL2 expression)

  • Compare with isotype controls to identify non-specific binding

• Validation Techniques:

  • Perform knockdown experiments (siRNA) targeting TKTL2 to confirm antibody specificity

  • Use Western blots to confirm single band of expected size (68 kDa)

  • Consider peptide competition assays where available synthetic peptides block specific binding

• Cross-Reactivity Assessment:

  • Review homology information: Antibodies targeting human TKTL2 may show cross-reactivity with other species based on sequence homology

  • BLAST analysis reveals varying degrees of homology: Human (100%), Mouse/Rat (92%), other mammals (84-92%)

  • Test antibodies on samples from multiple species if cross-species reactivity is desired

• Epitope Consideration:

  • Select antibodies targeting unique regions of TKTL2 to minimize cross-reactivity with related proteins (TKT, TKTL1)

  • N-terminal antibodies (aa 43-71) and those targeting aa 38-87 have shown good specificity

What factors influence reproducibility in TKTL2 expression analysis across different experimental platforms?

Factors affecting reproducibility in TKTL2 analysis include:

• Antibody Selection:

  • Monoclonal vs. polyclonal: Monoclonals offer higher reproducibility across experiments

  • Lot-to-lot variability: Use same antibody lot when possible for extended studies

  • Validation status: Prioritize antibodies with extensive validation data

• Sample Preparation Considerations:

  • Fixation methods for IHC: Overfixation or inadequate fixation affects epitope availability

  • Protein extraction methods for WB: Different lysis buffers may yield varying efficiency

  • Heat-mediated antigen retrieval is critical for successful IHC results

• Technical Execution:

  • Standardized protocols: Use consistent antibody concentrations, incubation times and temperatures

  • Scoring systems: For IHC, use validated scoring methods (intensity × percentage of positive cells)

  • Two independent observers should evaluate staining to minimize subjective interpretation

• Data Analysis:

  • Statistical methods: Apply appropriate statistical tests for data interpretation

  • Threshold definition: Consistent cut-offs for "high" vs "low" expression (e.g., IHC scores 0-3 as low and 4-15 as high)

  • Normalization methods: Use consistent reference genes/proteins for relative quantification

How might TKTL2 antibodies contribute to the development of targeted cancer therapies?

TKTL2 antibodies could advance targeted cancer therapies through:

• Companion Diagnostics:

  • Develop standardized IHC assays using TKTL2 antibodies to identify patients likely to respond to transketolase inhibitors

  • Stratify patients based on TKTL2 expression levels for clinical trials

  • Research shows high TKT expression associates with poor prognosis and could identify patients needing aggressive intervention

• Antibody-Drug Conjugates (ADCs):

  • Engineer TKTL2 antibodies conjugated to cytotoxic payloads

  • Target cancer cells with high TKTL2 expression while sparing normal tissues

  • Explore internalization kinetics of TKTL2 antibodies to optimize ADC design

• Combination Therapy Approaches:

  • Use TKTL2 antibodies to monitor expression during treatment with other therapies

  • Design rational combinations targeting both TKTL2 and interacting partners

  • Research has identified interactions with EGFR and MAPK3, suggesting potential synergy with inhibitors of these pathways

  • Studies show EGF treatment increases viability of TKT wild-type cells, which can be blocked by erlotinib (EGFR inhibitor)

What is the relationship between TKTL2 and other transketolase family members in cancer metabolism?

The relationship between TKTL2 and other transketolase family members reveals complex interactions:

• Functional Redundancy and Specificity:

  • TKT, TKTL1, and TKTL2 show differential expression patterns across cancer types

  • In lung adenocarcinoma, both TKT and TKTL2 are significantly upregulated while TKTL1 is expressed at low levels

  • This suggests non-redundant functions of family members in different cancers

• Comparative Expression Analysis:

  Transketolase Member	Expression in LUAD	Fold Change	P-value
  TKT	Upregulated	5.771	<0.001
  TKTL1	Low expression	0.197	<0.001
  TKTL2	Upregulated	0.025	<0.01

• Differential Prognostic Value:

  • High TKT expression significantly correlates with shorter OS (HR, 1.96; p=0.018) and DFS (HR, 1.77; p=0.04)

  • TKTL2 overexpression correlates with poor prognosis in ovarian cancer

  • This suggests family members may contribute to cancer progression through both overlapping and distinct mechanisms

• Metabolic Implications:

  • Inhibition of transketolase activity affects the pentose phosphate pathway

  • This has consequences for nucleotide synthesis and NADPH production

  • Understanding the specific contributions of each family member is crucial for targeted therapeutic approaches

How can multi-omics approaches incorporating TKTL2 antibodies enhance our understanding of cancer metabolism?

Multi-omics approaches with TKTL2 antibodies can reveal:

• Integrated Analysis Strategies:

  • Combine proteomics data (using TKTL2 antibodies) with transcriptomics and metabolomics

  • Use co-immunoprecipitation with TKTL2 antibodies followed by mass spectrometry to identify protein-protein interactions

  • Correlate TKTL2 expression with metabolic profiles to understand its impact on cancer metabolism

• Spatiotemporal Regulation Studies:

  • Analyze nuclear versus cytoplasmic localization of TKTL2 using immunofluorescence

  • Research shows nuclear transketolase promotes proliferation, viability, and migration in a non-metabolic manner

  • Identify factors governing subcellular localization and their impact on function

• TKTL2 Interactome Analysis:

  • Cross-linking Co-IP/MS analyses have revealed interactions with kinases and transcriptional coregulators

  • TKTL2 may interact with EGFR and MAPK3, associated with cell activation and stress response

  • These interactions suggest TKTL2 functions beyond its canonical metabolic role

  • Research has also connected TKTL2 with proteins like lactate dehydrogenase in cancer metabolism

• Single-Cell Analysis:

  • Use TKTL2 antibodies for single-cell proteomics or imaging mass cytometry

  • Identify heterogeneity in TKTL2 expression within tumors

  • Correlate with metabolic state and other cancer hallmarks at single-cell resolution

How does TKTL2 expression differ across cancer types and stages?

TKTL2 shows distinct expression patterns across cancer types:

What methodological approaches can resolve contradictory findings in TKTL2 research?

To address contradictions in TKTL2 research:

• Antibody Standardization:

  • Use well-characterized antibodies with validated specificity

  • Compare monoclonal and polyclonal antibodies targeting different epitopes

  • Document antibody validation methods including knockdown/knockout controls

  • Consider developing consensus-validated antibodies for standardized research

• Context-Dependent Analysis:

  • Systematically investigate cell-type and tissue-specific effects

  • Account for tumor microenvironment influences on TKTL2 function

  • Analyze both nuclear and cytoplasmic fractions separately to distinguish compartment-specific roles

• Comprehensive Mechanistic Studies:

  • Integrate metabolic and non-metabolic functions of TKTL2

  • Use both gain-of-function and loss-of-function approaches

  • Apply genome editing (CRISPR) for complete knockout versus RNAi for partial knockdown

  • Correlate in vitro findings with patient data using appropriate statistical methods

• Robust Statistical Analysis:

  • Use appropriate sample sizes with power calculations

  • Apply multivariate analysis to control for confounding factors

  • Standardize cut-off values for "high" versus "low" expression

  • Pre-register study designs to minimize reporting bias

How can researchers distinguish the functions of TKTL2 from other transketolase family members in experimental systems?

To distinguish TKTL2 functions from other family members:

• Specific Genetic Manipulation:

  • Design siRNAs targeting unique regions of TKTL2 mRNA

  • Use CRISPR-Cas9 to specifically knockout TKTL2 while leaving TKT and TKTL1 intact

  • Develop inducible systems for temporal control of TKTL2 expression

  • Create rescue experiments with TKTL2 mutants lacking specific domains

• Selective Antibody Approaches:

  • Use antibodies recognizing unique epitopes of TKTL2 not present in TKT or TKTL1

  • Validate antibody specificity through Western blots in cells with TKTL2 knockdown

  • Implement immunoprecipitation to isolate TKTL2-specific protein complexes

• Comparative Expression Analysis:

  • Simultaneously analyze all family members (TKT, TKTL1, TKTL2) in the same samples

  • Use multiplexed IHC or immunofluorescence to visualize co-expression patterns

  • Correlate with functional outcomes to determine individual contributions

• Differential Inhibitor Sensitivity:

  • Compare the effects of pan-transketolase inhibitors versus selective inhibitors

  • Design structure-based inhibitors exploiting unique features of TKTL2

  • Monitor metabolic fluxes to determine specific contributions to metabolic pathways