ptpru Antibody

Basic Research Questions

• What is PTPRU and why is it significant in research?

  PTPRU is a receptor-type protein tyrosine phosphatase that plays key roles in cell-cell recognition, adhesion, and signal transduction. It possesses an extracellular region containing a MAM domain, Ig-like and fibronectin type III-like repeats, along with a transmembrane region and two tandem intracellular catalytic domains . PTPRU has been implicated in neural development and cancer progression through dephosphorylation of proteins such as β-catenin. Research significance stems from its role in regulating various cellular processes including growth, differentiation, and oncogenic transformation .

• What are the recommended applications for PTPRU antibodies?

  PTPRU antibodies are primarily used in the following applications:

  Application	Recommended Dilutions	Notes
  Western Blot (WB)	1:500-1:2000	Detects isoforms around 130-200 kDa
  Immunohistochemistry (IHC)	1:50-1:500	Most effective with TE buffer pH 9.0 for antigen retrieval
  Immunofluorescence	As per manufacturer	Primarily detects nuclear localization in cancer cells
  ELISA	1:5000-1:20000	For highly sensitive detection

  Different antibodies may have varying optimal dilutions, so researchers should validate conditions for their specific experiments .

• How should researchers validate PTPRU antibodies before experimental use?

  Validation should follow multiple approaches to ensure specificity:

  • Compare expression in known positive and negative tissues or cell lines using Western blot

  • Include knockdown controls using shRNA or siRNA specifically targeting PTPRU

  • Validate subcellular localization by immunofluorescence and compare with cellular fractionation

  • For new antibodies, perform cross-reactivity testing against related PTP family members

  • Confirm specificity by using multiple antibodies raised against different epitopes of PTPRU

  In published studies, knockdown efficiency was verified by Western blot, immunofluorescence, and quantitative PCR to ensure antibody specificity .

• What are the key storage and handling considerations for PTPRU antibodies?

  Optimal preservation of PTPRU antibody activity requires careful handling:

  • Store most PTPRU antibodies at -20°C for long-term storage

  • For frequent use, aliquot and store at 4°C for up to one month

  • Most PTPRU antibodies are provided in buffered aqueous glycerol solutions with preservatives like sodium azide

  • Avoid repeated freeze-thaw cycles which can deteriorate antibody function

  • For reconstituted lyophilized antibodies, follow manufacturer's specifications (typically reconstitute in sterile PBS to 0.5 mg/mL)

  Storage conditions significantly impact antibody performance, making proper handling essential for experimental reproducibility.

• What cellular models are most appropriate for PTPRU antibody validation?

  Based on the literature, these models provide reliable systems for PTPRU research:

  • Human gastric cancer cell lines (AGS, SGC7901) show endogenous PTPRU expression

  • Human breast cancer cell line CAL51 exhibits endogenous PTPRU and is suitable for functional studies

  • HEK293T cells are effective for overexpression studies and validation of antibody specificity

  • Brain tissue samples show strong endogenous expression for immunohistochemical validation

  Cell models should be selected based on endogenous expression levels and relevance to the research question. PTPRU expression patterns vary across tissue types, with notable expression in neural tissues and certain cancer types .

Advanced Research Questions

• How can researchers distinguish between full-length PTPRU and its processed fragments in experimental systems?

  Distinguishing PTPRU isoforms requires strategic experimental approaches:

  • Use multiple antibodies targeting different epitopes: In gastric cancer cells, the 130kDa nuclear-localized PTPRU fragment is the predominant isoform, while full-length PTPRU (PTPRU-FL) is expressed at lower levels

  • Perform cellular fractionation: Nuclear and cytoplasmic protein fractions should be prepared using appropriate extraction kits to determine subcellular localization of different PTPRU forms

  • Compare band patterns with knockdown controls: PTPRU knockdown using validated shRNA helps identify which bands are specific

  • Cross-validate with antibodies raised against different domains: For example, antibodies directed against residues 850-950 (intracellular domain) detect a different pattern than antibodies against extracellular domains

  The PTPRU antibody in study detected the 130kDa nuclear fragment, while another antibody (PTPλ) targeting residues 850-950 detected both full-length PTPRU and a 120kDa isoform, confirming that different antibodies provide complementary information about PTPRU processing and localization.

• What methodological approaches are effective for studying PTPRU-dependent signaling pathways?

  Several validated approaches can reveal PTPRU's impact on signaling:

  • Knockdown experiments using lentivirus-delivered shRNA against PTPRU to assess effects on downstream signaling

  • Monitoring tyrosine phosphorylation status of β-catenin as a direct readout of PTPRU activity

  • Assessing nuclear translocation and transcriptional activity of β-catenin using reporter assays

  • Examining cell growth, migration, invasion, and adhesion as functional readouts

  • Using antibody-induced dimerization to modulate PTPRU activity and monitor effects on SRC phosphorylation

  In gastric cancer cells, PTPRU knockdown inhibited tyrosine phosphorylation and transcriptional activity of β-catenin while affecting levels of focal adhesion proteins and histone H3 lysine methylation, demonstrating PTPRU's multifaceted role in cellular signaling .

• How can antibodies be used to manipulate PTPRU function in experimental settings?

  Antibodies offer sophisticated tools for PTPRU functional modulation:

  • Monoclonal antibodies targeting PTPRU ectodomains can induce dimerization, inhibiting phosphatase activity

  • Antibody RD-43 has been shown to induce PTPRU dimer formation and promote degradation of the receptor

  • Size-exclusion chromatography with in-line multiangle light scattering (SEC-MALS) confirms antibody-PTPRU binding ratios

  • Coimmunoprecipitation assays can verify antibody-induced dimerization in cell models

  • Functional consequences can be monitored by examining downstream signaling effects, such as SRC inhibition

  Research has demonstrated that antibody-induced dimerization inhibits PTPRU activity prior to triggering receptor degradation, providing a time window to study the immediate effects of PTPRU inhibition separate from its degradation .

• What are the key differences in antibody selection and validation between PTPRU and other receptor tyrosine phosphatases?

  Important distinctions must be considered when working with different PTP family members:

  Receptor PTP	Molecular Weight	Key Applications	Epitope Considerations	Cross-reactivity Concerns
  PTPRU	130-200 kDa	WB, IHC, IF	Multiple isoforms; nuclear vs membrane	PTPRT, PTPRS, PTPRO
  PTPRS	140-217 kDa	WB, IHC	Full-length vs processed fragments	LAR family members
  PTPRT	164 kDa	IHC	Primarily in brain tissue	Type IIB receptor PTPs
  PTPRD	Variable	WB, IF	Extracellular domain targeting	LAR-like PTPs

  Cross-reactivity testing is essential, as demonstrated in where PTPRD antibodies were verified not to react with PTPRK, PTPRM, or PTPRT. Each PTP has distinct tissue expression patterns, with PTPRT primarily expressed in nervous system and PTPRU showing expression in various tissues including cancer cells .

• What approaches can resolve contradictory findings in PTPRU expression and function across different cancer types?

  Resolving contradictions requires systematic investigation:

  • Tissue-specific expression analysis: PTPRU appears to function as a tumor suppressor in colon cancer but is required for gastric cancer progression

  • Isoform-specific analysis: The 130kDa nuclear-localized PTPRU fragment is higher in gastric cancer tissues than adjacent non-cancer tissues

  • Careful knockdown studies: Knockdown of PTPRU in gastric cancer inhibited growth, migration, and invasion , opposite to its effects in other cancers

  • Context-dependent signaling: Examine PTPRU's effects on β-catenin signaling in different cellular contexts

  • Comprehensive subcellular localization: Nuclear vs. membrane localization may explain functional differences

  These contradictions suggest PTPRU may play context-dependent roles in different cancer types, potentially due to tissue-specific interaction partners or differential isoform expression.

• How can researchers optimize immunoprecipitation protocols for PTPRU antibodies to study protein-protein interactions?

  Effective immunoprecipitation requires:

  • Optimal lysis buffer: Use lysis buffer containing 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 1% NP-40, 150 mM NaCl, 1 mg/ml SDS, and 0.25 mg/ml sodium deoxycholate, supplemented with protease and phosphatase inhibitors

  • Antibody concentration: Use 4 μg/mL of PTPRU antibody for effective immunoprecipitation

  • Incubation conditions: Incubate cell lysate with antibody for 1 hour at 4°C followed by addition of Protein G magnetic beads

  • Washing protocol: Wash the beads three times with PBS for 15 minutes at room temperature

  • Validation controls: Include IgG controls and verify results using reciprocal immunoprecipitation with interacting partners

  For studying PTPRU dimers, co-transfection of differentially tagged PTPRU constructs (e.g., His- and V5-tagged) allows verification of dimer formation through co-immunoprecipitation experiments .

• What is the significance of PTPRU in quiescence and stem cell biology, and how can antibodies help elucidate these functions?

  PTPRU's role in quiescence presents a novel research avenue:

  • PTPRU is quiescence-induced in bone marrow mesenchymal stem cells (MSCs)

  • BrdU incorporation assays can be used in conjunction with PTPRU antibodies to assess proliferation status

  • Immunofluorescence with PTPRU antibodies can help track expression during cellular quiescence and reactivation

  • Functional studies using PTPRU antibodies may reveal its role in maintaining stem cell reserves

  • Comparison with other quiescence markers can establish PTPRU's position in regulatory networks

  This emerging area requires careful selection of antibodies that recognize the relevant isoforms expressed in stem cells and validation of specificity in these cellular contexts.

• How can researchers address the challenges of detecting low-abundance PTPRU in primary tissues?

  Several strategies can enhance detection sensitivity:

  • Signal amplification methods: Use enhanced chemiluminescence or tyramide signal amplification for Western blots

  • Optimized antigen retrieval: For IHC, use TE buffer at pH 9.0, which has been shown to improve detection

  • Enrichment techniques: Immunoprecipitation prior to Western blot can concentrate low-abundance proteins

  • Sensitivity testing: Use tissues with known high expression (brain) as positive controls

  • Multiple antibody approach: Use antibodies against different epitopes to confirm expression patterns

  • Verification with mRNA analysis: Complement protein detection with RT-qPCR to confirm expression

  These approaches are particularly important when studying PTPRU in non-cancerous tissues where expression levels may be lower than in cancer cell lines or tumor samples.