PHIP Antibody

What is PHIP and why is it important in cellular research?

PHIP (Pleckstrin Homology Domain Interacting Protein) functions as a probable regulator of insulin and insulin-like growth factor signaling pathways. Its significance stems from multiple cellular functions:

• Stimulates cell proliferation through regulation of cyclin transcription

• Demonstrates anti-apoptotic activity via AKT1 phosphorylation and activation

• Plays a crucial role in regulating cell morphology and cytoskeletal organization

• Binds to insulin receptor substrate 1 protein and regulates glucose transporter translocation in skeletal muscle cells

• May regulate growth and survival of pancreatic beta cells

Research has shown elevated copy numbers of PHIP may be associated with melanoma severity, suggesting its role in promoting melanoma metastasis in human patients, making it an important target for cancer research .

What are the key structural characteristics of commercially available PHIP antibodies?

Most commercially available PHIP antibodies target specific amino acid regions of the human PHIP protein. Analysis of current research antibodies reveals:

These antibodies are typically generated using recombinant fusion proteins or synthetic peptides corresponding to specific regions within human PHIP (NP_060404.3) .

What are the optimal applications for PHIP antibody detection methods?

PHIP antibodies have been validated across multiple detection platforms with varying efficacy. Based on current research protocols:

For experimental design, researchers should consider that WB generally provides the most consistent results across multiple cell lines, while IF offers better insight into subcellular localization patterns .

How should researchers optimize PHIP antibody protocols for challenging cell types?

When working with challenging cell types or tissues with low PHIP expression:

• Lysate preparation optimization: For difficult cell types, use RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors to prevent protein degradation.

• Signal enhancement strategies:

  • Increase antibody incubation time (overnight at 4°C)

  • Use signal amplification systems such as biotin-streptavidin

  • Consider fluorescent-conjugated antibodies (like CoraLite® Plus 488) for better detection in IF applications

• Antigen retrieval considerations: For formalin-fixed tissues, test both heat-induced epitope retrieval (citrate buffer, pH 6.0) and enzymatic retrieval methods to determine optimal conditions .

Always validate with positive control samples where PHIP expression has been confirmed (e.g., MCF-7, HepG2, A375 cell lines) .

How can researchers address common challenges with PHIP antibody specificity?

PHIP antibody studies frequently encounter specificity issues due to the protein's large size (207 kDa). To ensure robust results:

• Validation techniques for confirming specificity:

  • Always run positive controls from validated cell lines (HepG2, A375, MCF-7)

  • Include negative controls using PHIP knockdown/knockout samples

  • Confirm bands appear at the expected molecular weight (observed at ~207-230 kDa)

• Resolving multiple band detection:

  • Multiple bands may represent splice variants or post-translational modifications

  • Validate using peptide competition assays with the immunizing peptide

  • Consider testing multiple antibodies targeting different epitopes of PHIP

The observed molecular weight of PHIP can vary between 207-230 kDa due to post-translational modifications. Western blots typically show bands at approximately 230 kDa rather than the calculated 207 kDa .

What are the critical considerations when interpreting PHIP localization data?

Interpreting PHIP subcellular localization data requires careful consideration of:

• Fixation effects: Different fixation methods may affect epitope accessibility

  • Paraformaldehyde (4%) works well for most applications

  • Methanol fixation may better preserve certain epitopes

• Pattern interpretation:

  • PHIP typically shows both nuclear and cytoplasmic distribution

  • Nuclear localization may be cell-cycle dependent

  • Co-staining with markers for specific cellular compartments helps confirm localization

• Controls for accurate interpretation:

  • Include negative controls (secondary antibody only)

  • Use positive controls with known localization patterns

  • Consider co-staining with proteins known to interact with PHIP (e.g., insulin receptor substrates)

When contradictory localization data emerges, validate using complementary approaches like subcellular fractionation followed by Western blotting.

How can PHIP antibodies be utilized for studying protein-protein interactions?

For investigating PHIP interactions with binding partners:

• Co-immunoprecipitation (Co-IP) optimization:

  • Use gentle lysis buffers (e.g., NP-40 based) to preserve protein complexes

  • Optimal antibody amount: 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate

  • Pre-clear lysates to reduce non-specific binding

  • Consider crosslinking antibodies to beads to prevent IgG contamination

• Proximity ligation assay (PLA) approach:

  • Allows in situ detection of protein interactions

  • Requires two primary antibodies from different species

  • Can detect transient interactions missed by Co-IP

• ChIP applications:

  • PHIP antibodies have been successfully used in ChIP applications to study chromatin interactions

  • Use 5-10 μg antibody per ChIP reaction

  • Include appropriate controls (IgG, input DNA)

What strategies can researchers employ to study PHIP in the context of insulin signaling and cancer biology?

For investigating PHIP's role in insulin signaling and cancer:

• Insulin pathway activation studies:

  • Starve cells of serum (6-12 hours) followed by insulin stimulation

  • Monitor PHIP interactions with IRS-1 using co-IP

  • Analyze downstream AKT phosphorylation status

  • Use phospho-specific antibodies to monitor signaling events

• Cancer progression models:

  • Compare PHIP expression across cancer progression stages

  • Correlate with clinical outcomes in patient samples

  • Combine with markers for proliferation and metastasis

• Functional studies:

  • Use siRNA or CRISPR-Cas9 for PHIP knockdown/knockout

  • Rescue experiments with wild-type vs. mutant PHIP

  • Analyze effects on cell migration, invasion, and cell cycle progression

Research suggests PHIP may promote melanoma metastasis, making it a valuable target for cancer research applications.

How does PhIP-Seq technology differ from traditional PHIP antibody applications?

It's important to distinguish between PHIP antibodies and PhIP-Seq (Phage ImmunoPrecipitation Sequencing) technology:

PhIP-Seq uses phage-displayed peptide libraries to characterize antibody repertoires against numerous targets simultaneously, whereas PHIP antibodies specifically detect the PHIP protein in various experimental contexts .

What considerations should researchers make when designing longitudinal studies involving PHIP antibodies?

For longitudinal studies tracking PHIP in experimental models:

• Sample preservation strategies:

  • Flash-freezing tissues maintains epitope integrity

  • Consider OCT embedding for immunohistochemistry applications

  • Standardize fixation protocols across timepoints

• Antibody batch consistency:

  • Purchase sufficient antibody from single lot when possible

  • Include internal controls for normalization between experiments

  • Consider creating standard curves with recombinant PHIP

• Experimental controls for long-term studies:

  • Include time-matched controls for each experimental timepoint

  • Monitor antibody performance using positive control samples

  • Document any changes in reagents or protocols throughout study duration

• Quantitative analysis approaches:

  • Use digital image analysis software for consistent quantification

  • Develop standardized scoring systems for immunohistochemistry

  • Consider multiplexed approaches to analyze PHIP alongside other markers

When designing longitudinal studies, researchers should validate antibody performance across all anticipated experimental conditions before initiating long-term experiments.

What are the optimal storage conditions for maintaining PHIP antibody activity?

Proper storage significantly impacts PHIP antibody performance. Current research indicates:

• Temperature considerations:

  • Store at -20°C for long-term preservation

  • Avoid repeated freeze-thaw cycles; aliquot upon receipt

  • For conjugated antibodies (e.g., fluorescent-labeled), protect from light

• Buffer composition effects:

  • Most PHIP antibodies are stable in PBS with 0.02% sodium azide and 50% glycerol

  • Some formulations include 0.5% BSA for additional stability

  • Conjugated antibodies may contain additional stabilizers (e.g., Proclin300)

• Stability timeframes:

  • Unconjugated antibodies: Stable for one year after shipment at -20°C

  • Conjugated antibodies: Check expiration date; typically stable for 6-12 months

Small volume antibody preparations (20μl) often contain 0.1% BSA as a stabilizer, which should be considered when designing sensitive assays.

How should researchers validate PHIP antibody performance after extended storage?

To ensure reliable results with stored PHIP antibodies:

• Performance validation protocol:

  • Test with positive control samples (MCF-7, HepG2 cells)

  • Compare signal intensity with previous results

  • Check for increased background or non-specific binding

• Regeneration options for suboptimal performance:

  • Centrifuge to remove any aggregates (10,000g for 5 minutes)

  • Test a range of dilutions to re-optimize

  • Consider adding fresh protease inhibitors if degradation is suspected

• Documentation practices:

  • Record date of thawing and number of freeze-thaw cycles

  • Document lot number and initial validation results

  • Maintain positive control samples from initial validation for comparison