PTRH2 Antibody

What is PTRH2 and why is it important to study?

PTRH2 (Peptidyl-tRNA Hydrolase 2) is a bi-functional protein with critical roles in cellular processes including adhesion-mediated signaling, cell survival, and anoikis (programmed cell death due to loss of cell adhesion). PTRH2 modulates PI3K/AKT and ERK signaling pathways and regulates Bcl2 expression, making it central to cell growth, survival, and differentiation processes . It's particularly significant in two contrasting contexts: as a potential oncogene in cancer progression and as a critical developmental protein whose mutation leads to Infantile-onset Multisystem Nervous, Endocrine, and Pancreatic Disease (IMNEPD) .

What types of PTRH2 antibodies are available for research?

Multiple PTRH2 antibodies are available with varying specifications:

Most commercially available PTRH2 antibodies are rabbit polyclonal antibodies with applications in Western blotting and immunohistochemistry .

How should I store and handle PTRH2 antibodies?

Most PTRH2 antibodies should be stored at -20°C . Many are supplied in a buffer containing glycerol (typically 40-50%) and sodium azide (0.02-0.05%) to maintain stability and prevent microbial growth . For optimal performance:

• Avoid repeated freeze-thaw cycles by aliquoting the antibody

• Keep on ice during experimental procedures

• Return to -20°C immediately after use

• Follow manufacturer-specific storage recommendations for each antibody

• Note that some formulations (like those with high glycerol content) may not require aliquoting

What are the appropriate dilutions for different applications of PTRH2 antibodies?

Dilution requirements vary by application and specific antibody:

For example, antibody 51006-2-AP has recommended dilutions of 1:500-1:3000 for WB, 1:50-1:500 for IHC, and 1:20-1:200 for IF/ICC . Always titrate the antibody in your specific experimental system for optimal results.

How should I design Western blot experiments to detect PTRH2?

For optimal Western blot detection of PTRH2:

• Sample preparation:

  • Use appropriate lysis buffers with protease inhibitors

  • Load 20-40μg of total protein per lane

• Gel selection:

  • Use 12% SDS-PAGE gels, as PTRH2 has a molecular weight of approximately 19 kDa

• Antibody selection and dilution:

  • Primary antibody: Start with 1:1000 dilution and adjust based on signal intensity

  • Secondary antibody: Use appropriate anti-rabbit IgG conjugated to HRP at recommended dilution

• Controls:

  • Include positive control samples (e.g., HepG2, HEK-293, Jurkat, MCF-7, or Raji cells)

  • Consider using PTRH2 knockout/knockdown samples as negative controls

• Expected results:

  • Look for a band at approximately 19 kDa, which corresponds to the PTRH2 protein

What are the optimal methods for immunohistochemical detection of PTRH2?

For IHC detection of PTRH2:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections

  • For PTRH2, successful staining has been reported in human prostate cancer tissue

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Blocking and antibody incubation:

  • Use appropriate blocking solution (e.g., 5% normal goat serum)

  • Incubate with primary antibody at 1:50-1:500 dilution

  • Use appropriate detection system (e.g., HRP-conjugated secondary antibody and DAB)

• Controls and validation:

  • Include positive control tissues with known PTRH2 expression

  • Use isotype control antibodies to assess non-specific binding

• Interpretation:

  • PTRH2 staining may be observed in multiple subcellular locations due to its translocation between mitochondria and cytoplasm

How can I investigate PTRH2's dual role in cell survival and anoikis?

To investigate PTRH2's context-dependent functions:

• Adhesion vs. suspension culture experiments:

  • Compare PTRH2 localization and function in adherent vs. suspended cells using immunofluorescence

  • Track translocation from mitochondria to cytoplasm during detachment-induced anoikis

• Protein interaction studies:

  • Use co-immunoprecipitation to detect PTRH2 interaction with Gro/Tle transcriptional co-repressor during anoikis

  • Investigate PTRH2's effect on Bcl2 expression through transcriptional regulation

• Signaling pathway analysis:

  • Examine PI3K/AKT and ERK pathway activation states in relation to PTRH2 expression

  • Use pathway inhibitors in combination with PTRH2 modulation to establish hierarchical relationships

• Functional assays:

  • Measure anoikis resistance in PTRH2 overexpression and knockdown models

  • Assess cell survival and proliferation in adherent vs. suspended culture conditions

How can I address antibody cross-reactivity or specificity concerns with PTRH2 antibodies?

To validate PTRH2 antibody specificity:

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of PTRH2 (e.g., N-terminal vs. C-terminal)

  • Compare results from antibodies from different vendors or different clones

• Knockout/knockdown validation:

  • Use CRISPR/Cas9-mediated PTRH2 knockout or siRNA knockdown cells as negative controls

  • Confirm disappearance of signal in these models

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide before application

  • Signal should be blocked or significantly reduced if antibody is specific

• Recombinant protein expression:

  • Express tagged PTRH2 (e.g., with FLAG or His tag) in cells

  • Compare detection using anti-PTRH2 antibody versus anti-tag antibody

• Mass spectrometry validation:

  • Confirm identity of immunoprecipitated band by mass spectrometry analysis

What are the best approaches to study PTRH2 in the context of its role in cancer?

To investigate PTRH2's oncogenic potential:

• Expression analysis:

  • Compare PTRH2 levels in tumor vs. normal tissues using IHC and Western blotting

  • Correlate expression with clinical parameters and patient outcomes using tissue microarrays

• Functional studies:

  • Conduct gain/loss-of-function experiments in cancer cell lines

  • Assess effects on proliferation, migration, invasion, and anoikis resistance

  • Use 3D culture models to better recapitulate tumor microenvironment

• Signaling pathway investigation:

  • Examine PI3K/AKT and ERK pathway activation in response to PTRH2 modulation

  • Measure Bcl2 expression levels and correlation with PTRH2 expression

• In vivo models:

  • Develop xenograft models with PTRH2 overexpression or knockdown

  • Assess tumor growth, metastasis, and response to therapies

• Drug resistance studies:

  • Investigate PTRH2's potential role in resistance to apoptosis-inducing chemotherapeutics

  • Test combination approaches targeting PTRH2-mediated survival pathways

What are common issues with PTRH2 antibody staining and how can I overcome them?

Common issues and solutions:

• High background in Western blots:

  • Increase blocking time/concentration

  • Use different blocking agents (BSA vs. milk)

  • Increase washing steps duration and number

  • Reduce primary antibody concentration

  • Test different antibody diluents

• Weak or no signal in IHC:

  • Optimize antigen retrieval (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Increase antibody concentration or incubation time

  • Use signal amplification systems

  • Ensure tissue is properly fixed and processed

  • Confirm PTRH2 expression in your tissue of interest

• Multiple bands in Western blot:

  • Verify expected molecular weight (19 kDa for PTRH2)

  • Test specificity using blocking peptides

  • Optimize sample preparation to reduce protein degradation

  • Use fresh antibody aliquots to avoid degradation

• Inconsistent immunofluorescence results:

  • Standardize fixation protocols

  • Optimize permeabilization conditions for mitochondrial and cytoplasmic detection

  • Use confocal microscopy to better visualize subcellular localization

How should I interpret changes in PTRH2 localization between different cellular compartments?

PTRH2 localization is functionally significant and requires careful interpretation:

• Mitochondrial localization:

  • Typically indicates normal cellular state in adherent cells

  • Confirm with mitochondrial markers (e.g., MitoTracker, TOMM20)

• Cytoplasmic translocation:

  • Often observed during anoikis or loss of cell adhesion

  • Associated with increased interaction with Gro/Tle transcriptional co-repressors

  • May indicate active pro-apoptotic function

• Quantification methods:

  • Use cellular fractionation followed by Western blotting

  • Employ high-resolution confocal microscopy with colocalization analysis

  • Calculate nuclear/cytoplasmic or mitochondrial/cytoplasmic ratio

• Context-dependent interpretation:

  • In cancer cells, altered localization patterns may indicate pathological changes

  • In developmental contexts, localization may correlate with differentiation states

How can I integrate PTRH2 antibody data with functional studies to understand disease mechanisms?

For comprehensive mechanistic studies:

• Correlative approaches:

  • Link PTRH2 expression patterns with cellular phenotypes

  • Correlate expression/localization with activation of downstream pathways (PI3K/AKT, ERK)

  • Relate PTRH2 levels to Bcl2 expression and cell survival outcomes

• Genetic models:

  • Study PTRH2 mutations identified in IMNEPD patients

  • Create cellular models expressing these mutations

  • Compare antibody-based detection with functional outcomes

• Multi-omics integration:

  • Combine antibody-based protein detection with transcriptomics data

  • Correlate protein localization/expression with phosphoproteomics

  • Integrate with interactome data to build comprehensive signaling networks

• Therapeutic development context:

  • Use antibody-based assays to screen for compounds that modulate PTRH2 functions

  • Develop biomarker strategies based on PTRH2 expression patterns

  • Monitor treatment responses using PTRH2 as a readout

How can PTRH2 antibodies be used to study the protein's role in developmental disorders?

For investigating PTRH2 in IMNEPD and developmental contexts:

• Patient sample analysis:

  • Compare PTRH2 expression, localization, and function in patient vs. control samples

  • Use antibodies to detect mutant forms and potential changes in localization or stability

• Developmental timing studies:

  • Track PTRH2 expression during normal development using stage-specific tissues

  • Correlate with muscle differentiation markers and caspase-3 activation

• Tissue-specific investigations:

  • Focus on affected tissues (skeletal muscle, nervous system, endocrine system, pancreas)

  • Compare PTRH2 expression patterns across these tissues

• Model systems:

  • Develop animal models with PTRH2 mutations

  • Use antibodies to validate expression changes and phenocopy human disease

• Therapeutic monitoring:

  • Assess potential therapies by monitoring PTRH2 expression/function restoration

  • Use antibody-based assays as readouts for intervention efficacy

What are the technical considerations for developing co-localization assays for PTRH2?

For advanced co-localization studies:

• Antibody compatibility:

  • Select antibodies raised in different host species to allow co-staining

  • Ensure primary antibodies are compatible with your experimental system

• Subcellular markers:

  • For mitochondrial co-localization: Use established markers like TOMM20, COX4, or MitoTracker dyes

  • For cytoplasmic studies: Use cytoskeletal markers or cytoplasmic proteins

• Imaging considerations:

  • Use confocal or super-resolution microscopy to accurately assess co-localization

  • Employ Z-stack imaging to capture the full cellular volume

  • Consider live-cell imaging to track dynamic PTRH2 translocation events

• Quantitative analysis:

  • Calculate Pearson's or Mander's co-localization coefficients

  • Use specialized software (ImageJ with JACoP plugin, CellProfiler, etc.)

  • Develop threshold-based masking for specific compartments

• Controls:

  • Include positive controls (known interacting proteins)

  • Use negative controls (proteins known not to interact or co-localize)

  • Validate findings with biochemical approaches (co-IP, proximity ligation assay)

How can I apply PTRH2 antibodies in high-throughput or multiplexed analyses?

For advanced screening and multiplexed approaches:

• Tissue microarray analysis:

  • Apply PTRH2 antibodies to tissue microarrays for rapid multi-sample analysis

  • Correlate expression with clinicopathological parameters

• Multiplexed immunofluorescence:

  • Use spectral unmixing systems to detect multiple markers alongside PTRH2

  • Apply cyclic immunofluorescence methods for high-dimensional data

• High-content screening:

  • Develop cell-based assays measuring PTRH2 expression, localization, and downstream effects

  • Screen compounds that modulate PTRH2 functions or restore mutant protein activity

• Flow cytometry applications:

  • Optimize intracellular staining protocols for PTRH2

  • Combine with cell surface markers and functional readouts

• Mass cytometry (CyTOF):

  • Develop metal-conjugated PTRH2 antibodies for high-dimensional analysis

  • Integrate with other signaling pathway markers