TPD52L2 Antibody, HRP conjugated

What is TPD52L2 and why is it targeted in research?

TPD52L2, also known as tumor protein D52-like 2 or D54, is a small coiled-coil motif bearing protein that plays significant roles in cellular processes. Research has demonstrated its involvement in the proliferation, invasiveness, and apoptosis of cancer cells, particularly in glioblastoma through modulation of Wnt/β-catenin/Snail signaling pathways . Studies have also identified TPD52L2 autoantibodies in meningioma patients, suggesting its potential as a biomarker . The targeting of TPD52L2 in research settings allows for the investigation of cancer pathways and potential therapeutic interventions.

What are the recommended applications for TPD52L2 antibodies?

TPD52L2 antibodies have been validated for multiple applications with specific recommended dilutions:

Researchers should note that antibody performance may be sample-dependent, and titration is recommended in each testing system to obtain optimal results .

How do I choose between polyclonal and monoclonal TPD52L2 antibodies?

When selecting between polyclonal and monoclonal TPD52L2 antibodies, consider your specific research needs:

Polyclonal antibodies (such as the rabbit polyclonal 11795-1-AP or goat polyclonal A83448) recognize multiple epitopes on the TPD52L2 protein, providing stronger signals in applications like Western blot and IHC. They are particularly useful for detecting proteins expressed at low levels or for initial screening .

Monoclonal antibodies offer higher specificity to a single epitope, reducing background and cross-reactivity. They are preferred for studies requiring absolute specificity or when investigating subtle protein modifications.

For applications involving HRP detection systems, both antibody types can be used with appropriate secondary antibodies. The selection should be based on the sensitivity and specificity requirements of your particular experimental design.

How should I optimize Western blotting protocols for TPD52L2 detection with HRP-conjugated systems?

For optimal detection of TPD52L2 using Western blot with HRP-conjugated systems:

• Sample preparation: Use RIPA buffer for cell lysis with protease inhibitors. Load 25-35μg of protein per lane, as validated in human breast cancer lysate studies .

• Gel selection: Use 10-12% SDS-PAGE gels for optimal separation of TPD52L2 (~22-30 kDa).

• Primary antibody incubation: Dilute unconjugated TPD52L2 antibody to 1:500-1:1000 (11795-1-AP) or 0.1-0.3μg/ml (A83448) in 5% BSA/TBST and incubate overnight at 4°C .

• Secondary antibody selection: For HRP detection, use:

  • Anti-rabbit HRP (1:5000) if using rabbit primary antibodies

  • Anti-goat HRP (1:5000) if using goat primary antibodies such as A83448

• Signal detection: Use enhanced chemiluminescence for visualization. Expected molecular weight of TPD52L2 is 25-30 kDa, slightly higher than the calculated 22 kDa, possibly due to post-translational modifications .

• Controls: Include positive controls such as HEK-293 cells, MCF-7 cells, or brain tissue samples which have been validated to express TPD52L2 .

What are the critical factors when performing immunohistochemistry for TPD52L2 in cancer tissues?

When performing IHC for TPD52L2 in cancer tissues, consider these critical factors:

• Tissue preparation: Use formalin-fixed paraffin-embedded (FFPE) tissue sections at 4-6μm thickness.

• Antigen retrieval: This is crucial for TPD52L2 detection. Use TE buffer pH 9.0 as the primary recommended method. Alternatively, citrate buffer pH 6.0 can be used, though potentially with reduced efficiency .

• Antibody dilution: Use TPD52L2 antibodies at 1:50-1:500 for IHC applications. For A83448, a concentration of 3.75μg/ml has been validated .

• Detection system: For HRP-based detection systems:

  • Use HRP-conjugated secondary antibodies appropriate for your primary antibody host species

  • Develop with DAB substrate for a brown reaction product

  • Counterstain with hematoxylin for nuclear visualization

• Positive control selection: Include human breast cancer tissue as a positive control, which has been validated for TPD52L2 expression .

• Negative controls: Include sections with no primary antibody and tissue known to be negative for TPD52L2 to assess background staining.

• Scoring system: Develop a consistent scoring system for TPD52L2 expression, considering both staining intensity and percentage of positive cells.

How can TPD52L2 antibodies be used to investigate its role in cancer cell invasion and migration?

TPD52L2 has been identified as a regulator of cell invasion in glioblastoma, with downregulation enhancing invasion while inhibiting cell proliferation . To investigate this role:

• Knockdown/Overexpression studies: Use TPD52L2 antibodies to verify protein expression levels following genetic manipulation (siRNA, CRISPR-Cas9, or overexpression vectors).

• Invasion assays: Employ transwell or Boyden chamber assays to quantify invasive capacity following TPD52L2 modulation, then use Western blotting with TPD52L2 antibodies to correlate protein expression with phenotype.

• Signaling pathway analysis: Investigate the Wnt/β-catenin/Snail pathway components (particularly CTNNB1/β-catenin and SNAI1/Snail) using Western blot or immunofluorescence to determine how TPD52L2 levels affect EMT (epithelial-mesenchymal transition) process markers .

• Morphological studies: Examine cellular protrusion formation, as research has shown cells with longer protrusions exhibit higher invasive ability and lower sensitivity to treatment. Use immunofluorescence with TPD52L2 antibodies alongside cytoskeletal markers .

• In vivo models: Utilize animal models with TPD52L2-modulated cancer cells and employ IHC with TPD52L2 antibodies to assess protein expression in tumors and correlate with invasive phenotypes.

For detection in these applications, HRP-conjugated secondary antibodies provide sensitive visualization in Western blots and IHC, while fluorescent secondaries are preferable for co-localization studies in immunofluorescence.

What methods can be used to study TPD52L2 as a potential biomarker in different cancer types?

Research has identified TPD52L2 as a potential biomarker in cancers including glioblastoma and meningioma . To effectively study its biomarker potential:

• Tissue microarray (TMA) analysis: Use TPD52L2 antibodies for IHC on TMAs containing multiple patient samples to assess expression patterns across tumor grades and types. Correlate expression with clinical outcomes as demonstrated in the study of 88 GBM cases, where low TPD52L2 expression was associated with worse prognosis in patients receiving the Stupp treatment strategy .

• Autoantibody detection: Analyze patient sera for TPD52L2 autoantibodies using ELISA or protein arrays, as demonstrated in meningioma studies where TPD52L2 was identified as one of 25 proteins showing both differential expression and autoantibody responses .

• Multi-omics integration:

  • Compare antibody-based protein detection with mass spectrometry data

  • Correlate with transcriptomics data to identify discrepancies between mRNA and protein levels

  • Integrate with patient outcome data to establish clinical relevance

• Liquid biopsy development: Investigate TPD52L2 presence in circulating tumor cells or exosomes using immunoprecipitation followed by Western blot with HRP-conjugated detection systems.

• Correlation with treatment response: Use TPD52L2 antibodies to assess protein levels before and after treatment, correlating with patient outcomes to determine predictive value. Research has shown that TPD52L2 levels impact sensitivity to temozolomide and radiation in glioblastoma .

How can phospho-specific TPD52L2 antibodies contribute to understanding its regulation?

While the provided search results don't specifically mention phospho-specific TPD52L2 antibodies, this represents an important advanced research direction:

• Identification of phosphorylation sites: Use mass spectrometry data to identify potential phosphorylation sites on TPD52L2 before developing or selecting phospho-specific antibodies.

• Validation strategies for phospho-specific antibodies:

  • Western blot comparing phosphatase-treated versus untreated samples

  • Immunoprecipitation followed by phospho-protein staining

  • Peptide competition assays with phosphorylated versus non-phosphorylated peptides

• Signaling pathway analysis: Use phospho-specific TPD52L2 antibodies alongside antibodies for kinases in the Wnt/β-catenin pathway to determine regulation mechanisms and activation triggers.

• Temporal dynamics: Study phosphorylation changes in response to stimuli using time-course experiments with phospho-specific antibodies and HRP-conjugated detection systems.

• Correlation with function: Determine how phosphorylation status affects TPD52L2 function in proliferation and invasion by correlating phospho-specific antibody signals with cellular phenotypes.

What are the most common issues when using TPD52L2 antibodies and how can they be resolved?

When working with TPD52L2 antibodies, researchers may encounter these common challenges:

• Multiple bands in Western blot:

  • Issue: Observing bands outside the expected 25-30 kDa range.

  • Solution: Optimize primary antibody concentration (start with 1:500-1:1000 dilution); increase blocking time/concentration; include appropriate controls including knockout/knockdown samples if available .

• Weak signal in IHC/IF:

  • Issue: Poor or no staining despite proper controls.

  • Solution: Ensure proper antigen retrieval (use recommended TE buffer pH 9.0); try higher antibody concentration; extend primary antibody incubation time; apply signal amplification systems like polymer-HRP detection .

• High background:

  • Issue: Non-specific staining obscuring true signal.

  • Solution: Increase blocking time; use more stringent wash conditions; decrease primary antibody concentration; ensure secondary antibody compatibility.

• Inconsistent results between experiments:

  • Issue: Variable signal intensity or localization between replicates.

  • Solution: Standardize protocols; use consistent lot numbers; prepare fresh working solutions; maintain consistent incubation times and temperatures.

• Discrepancy between techniques:

  • Issue: TPD52L2 detected by one method but not another.

  • Solution: Each technique detects proteins in different states; consider native vs. denatured conditions; examine epitope accessibility in different applications; validate with multiple antibodies targeting different epitopes.

How should samples be prepared to maximize TPD52L2 detection in different experimental systems?

Optimal sample preparation is crucial for reliable TPD52L2 detection:

• Cell/Tissue lysis for Western blot:

  • Use RIPA buffer with protease inhibitors for most applications

  • For detecting protein interactions, consider milder NP-40 or Triton X-100 buffers

  • Prepare fresh lysates or store at -80°C with minimal freeze-thaw cycles

  • Sonicate briefly to shear DNA and reduce sample viscosity

• Tissue preparation for IHC:

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

  • Ensure consistent fixation time to prevent overfixation

  • Process and embed in paraffin following standard protocols

  • Cut sections at 4-6μm thickness

  • For TPD52L2, perform antigen retrieval with TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• Cell preparation for IF/ICC:

  • Culture cells on appropriate slides or coverslips

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

  • Permeabilize with 0.1% Triton X-100 for intracellular proteins

  • Block thoroughly to reduce background

  • HepG2 cells have been validated as positive controls for TPD52L2 immunofluorescence

• Protein lysate for IP:

  • Use 1.0-3.0 mg of total protein lysate with 0.5-4.0 μg of TPD52L2 antibody

  • HEK-293 cells have been validated for successful TPD52L2 immunoprecipitation

  • Include appropriate controls (IgG, input, non-specific binding)

• Sample amounts for different cell types:

  • For Western blot, validated positive samples include HEK-293 cells, mouse/rat brain tissue, and MCF-7 cells

  • Loading 25-35μg of total protein per lane is recommended based on validation studies

How can TPD52L2 antibodies be integrated into single-cell analysis techniques?

Integrating TPD52L2 antibodies into single-cell analysis represents an advanced application that can reveal cellular heterogeneity in tumor samples:

• Mass cytometry (CyTOF):

  • Conjugate TPD52L2 antibodies with rare metal isotopes

  • Combine with other cancer markers for multiplexed phenotyping

  • Correlate TPD52L2 expression with cell subpopulations and functional states

  • Particularly relevant given findings on cellular heterogeneity in glioblastoma where single clone cultures exhibited distinct biological phenotypes

• Single-cell Western blotting:

  • Apply microfluidic platforms for protein analysis at single-cell resolution

  • Use TPD52L2 antibodies with HRP-conjugated detection systems

  • Quantify expression variability across individual tumor cells

  • Correlate with morphological features like cellular protrusions that have been linked to invasiveness

• Imaging mass cytometry:

  • Perform highly multiplexed imaging of tissue sections

  • Include TPD52L2 antibodies to correlate spatial distribution with other markers

  • Study tumor heterogeneity and microenvironment interactions

  • Particularly valuable given the observed heterogeneity between tumor margins and core lesions in glioblastoma

• Flow cytometry applications:

  • Optimize intracellular staining protocols for TPD52L2

  • Use with surface markers to identify specific cell populations

  • Sort cells based on TPD52L2 expression for downstream functional assays

  • Investigate correlation between expression levels and functional phenotypes like invasion capacity

• Spatial transcriptomics correlation:

  • Combine TPD52L2 antibody-based imaging with spatial transcriptomics

  • Correlate protein expression with mRNA levels at single-cell resolution

  • Investigate post-transcriptional regulation mechanisms

What is the significance of TPD52L2 in relation to cancer treatment resistance mechanisms?

Research has demonstrated that TPD52L2 expression levels impact cancer treatment responses, particularly in glioblastoma:

• Chemotherapy resistance:

  • Downregulation of TPD52L2 was found to reduce sensitivity to temozolomide (TMZ), a standard chemotherapy agent for glioblastoma

  • GBM patients with low TPD52L2 expression showed worse prognosis when treated with the standard Stupp strategy (radiotherapy plus TMZ)

  • To study this mechanism, researchers can use TPD52L2 antibodies to:

    • Monitor protein levels before and after treatment

    • Correlate expression with patient outcomes

    • Examine associated signaling pathways using co-immunoprecipitation or proximity ligation assays

• Radiation resistance:

  • TPD52L2 levels affect sensitivity to radiation therapy in GBM

  • Cells with longer protrusions (associated with lower TPD52L2) showed reduced radiation sensitivity

  • Research applications include:

    • Using TPD52L2 antibodies to characterize resistant cell populations

    • Developing TPD52L2-based predictive biomarkers for treatment response

    • Investigating combinatorial approaches to overcome resistance

• Wnt/β-catenin pathway involvement:

  • TPD52L2 modulates the Wnt/β-catenin/Snail signaling pathway

  • This pathway is implicated in treatment resistance across multiple cancer types

  • Research strategies include:

    • Using TPD52L2 antibodies alongside Wnt pathway component antibodies

    • Correlating pathway activation with treatment outcomes

    • Investigating potential for combination therapies targeting both TPD52L2 and Wnt signaling

• Clinical applications:

  • Development of TPD52L2-based companion diagnostics for treatment selection

  • Stratification of patients for clinical trials based on TPD52L2 expression

  • Investigation of TPD52L2 as a therapeutic target to overcome resistance