INPP5B Antibody, HRP conjugated

What is INPP5B and why is it an important research target?

INPP5B is a type II inositol polyphosphate 5-phosphatase that preferentially hydrolyzes the 5-phosphate of both phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3]. It also displays activity towards soluble inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5)P4] . INPP5B has recently been identified as a key regulator of actin remodeling, B cell receptor (BCR) clustering, and downstream signaling in antigen-stimulated B cells, making it a potential therapeutic target for B cell malignancies caused by aberrant BCR signaling . Its structural similarity to OCRL1 (the protein mutated in Lowe syndrome) and its ability to compensate for OCRL1 loss in knockout mice further highlight its biological significance .

What applications are most suitable for HRP-conjugated INPP5B antibodies?

HRP-conjugated INPP5B antibodies are particularly valuable for applications requiring signal amplification and colorimetric or chemiluminescent detection. These include Western blotting, immunohistochemistry on paraffin-embedded sections (IHC-P), and enzyme immunoassays (EIA) . The HRP conjugation eliminates the need for secondary antibody incubation, reducing background signal and experimental time. For optimal results in Western blotting, use dilutions between 1:1000-1:5000, depending on protein expression levels and detection system sensitivity. In immunohistochemistry, dilutions of 1:100-1:500 are typically effective, while EIAs may require optimization between 1:500-1:2000 depending on your specific assay format.

How should researchers validate INPP5B antibody specificity before experimental use?

Thorough validation is essential given the structural similarity between INPP5B and other inositol phosphatases, particularly OCRL1 which shares 45% sequence identity and similar domain architecture . A comprehensive validation approach should include:

• Western blot analysis using both positive controls (tissues/cells known to express INPP5B) and negative controls (knockout or knockdown systems)

• Peptide competition assays using the immunizing peptide (typically from C-terminal region, AA 957-987 for many commercial antibodies)

• Cross-reactivity testing against recombinant OCRL1 and other related phosphatases

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Parallel testing with multiple antibodies targeting different INPP5B epitopes

Validation results should demonstrate a single band at approximately 75kDa, corresponding to full-length INPP5B, with minimal cross-reactivity to related proteins.

What subcellular compartments should researchers focus on when studying INPP5B localization?

When designing experiments to study INPP5B localization, researchers should focus on both the early secretory pathway and endocytic compartments. INPP5B has been localized to the Golgi apparatus and ER-to-Golgi intermediate compartment (ERGIC), with better overlap with cis-Golgi markers (GM130) than trans-Golgi markers (TGN46) . There is also evidence for INPP5B association with enlarged endosomes upon expression of constitutively active RAB5 and with growth-factor-induced plasma membrane ruffles . Unlike its close homolog OCRL1, INPP5B does not significantly associate with clathrin-coated intermediates. For comprehensive localization studies, researchers should employ co-staining with markers for:

• cis-Golgi (GM130)

• ERGIC (ERGIC53)

• trans-Golgi network (TGN46)

• Early endosomes (EEA1, RAB5)

• Plasma membrane ruffles (following growth factor stimulation)

This approach will enable researchers to accurately track INPP5B's distribution and potential redistribution under various experimental conditions.

How can researchers effectively design experiments to study the role of INPP5B in B cell receptor signaling?

Based on recent findings that INPP5B regulates actin remodeling and BCR clustering , researchers investigating INPP5B in BCR signaling should design experiments that capture both morphological changes and downstream signaling events. A comprehensive experimental design should include:

• Temporal analysis of BCR clustering:

  • Use fluorescently labeled monovalent anti-IgM Fab fragments to visualize BCR

  • Apply TIRF microscopy to monitor cluster formation in real-time

  • Quantify cluster number, size, and intensity over time following stimulation

• Assessment of actin remodeling:

  • Co-stain with phalloidin to visualize F-actin reorganization

  • Measure cell spreading on antibody-coated surfaces

  • Correlate BCR clustering with actin cytoskeletal changes

• Downstream signaling evaluation:

  • Monitor phosphorylation of key signaling proteins (e.g., SYK, BTK, PLCγ2)

  • Assess calcium mobilization using fluorescent indicators

  • Measure activation of transcription factors like NF-κB and NFAT

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated knockout or knockdown

  • Rescue experiments with wild-type vs. catalytically inactive INPP5B

  • Acute protein depletion using auxin-inducible degron systems

This multi-faceted approach will provide comprehensive insights into INPP5B's role in coordinating BCR signaling and cytoskeletal reorganization.

What controls should be included when using HRP-conjugated INPP5B antibodies for immunohistochemistry?

A robust immunohistochemistry protocol using HRP-conjugated INPP5B antibodies requires comprehensive controls to ensure specificity and reliability:

• Positive tissue controls:

  • Include tissues known to express INPP5B (e.g., liver, kidney)

  • Process these tissues identically to experimental samples

• Negative tissue controls:

  • Include tissues with minimal INPP5B expression

  • Consider INPP5B knockout tissues when available

• Technical controls:

  • Omission of primary antibody (to assess non-specific binding of detection systems)

  • Isotype control (matching host species and immunoglobulin class)

  • Absorption control (pre-incubation with the immunizing peptide)

  • Concentration gradients (to determine optimal antibody dilution)

• Signal validation controls:

  • Parallel staining with unconjugated antibody followed by HRP-conjugated secondary

  • Comparison with alternative detection methods (e.g., fluorescence)

  • Correlation with mRNA expression (e.g., by ISH or qPCR on adjacent sections)

• Endogenous peroxidase blocking verification:

  • Include tissue sections treated only with HRP substrate to confirm complete blocking

These controls will help distinguish genuine INPP5B immunoreactivity from technical artifacts, ensuring reliable and reproducible results.

How can researchers investigate INPP5B interactions with RAB proteins in the secretory pathway?

INPP5B binds multiple RAB proteins in the secretory pathway, including RAB1A and RAB2A (ERGIC/cis-Golgi), RAB33B and RAB6A (Golgi stack), RAB8A and RAB9A . To comprehensively study these interactions, researchers should employ a multi-methodological approach:

• In vitro binding assays:

  • GST-pulldown assays using purified GST-RAB proteins loaded with GTPγS (active) or GDP (inactive)

  • Surface plasmon resonance (SPR) to determine binding kinetics and affinities

  • Isothermal titration calorimetry (ITC) for thermodynamic characterization

• Cellular interaction studies:

  • Co-immunoprecipitation with antibodies against endogenous proteins

  • Proximity ligation assay (PLA) to visualize interactions in situ

  • FRET or BiFC to monitor interactions in living cells

• Structural characterization:

  • X-ray crystallography of INPP5B-RAB complexes

  • Cryo-EM for larger assemblies

  • NMR for mapping interaction interfaces

• Functional validation:

  • Mutagenesis of putative RAB-binding regions in INPP5B

  • Expression of RAB-binding deficient mutants to assess effects on INPP5B localization

  • Dominant-negative RAB expression to disrupt specific trafficking steps

This comprehensive approach will provide detailed insights into how INPP5B is targeted to specific membrane compartments through RAB interactions and how these interactions regulate INPP5B function in membrane trafficking pathways.

What methodologies can differentiate between INPP5B and OCRL1 functions despite their structural similarities?

Distinguishing the functions of INPP5B from its close homolog OCRL1 requires sophisticated experimental approaches that account for their structural similarities (45% sequence identity) while exploiting their differences :

• Comparative interaction proteomics:

  • BioID or APEX proximity labeling with INPP5B vs. OCRL1 as baits

  • Quantitative immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening with specific domains from each protein

• Domain-swapping experiments:

  • Create chimeric proteins with swapped domains between INPP5B and OCRL1

  • Assess localization, binding partners, and functional outcomes

  • Focus on the clathrin-binding region present in OCRL1 but absent in INPP5B

• Acute and conditional depletion strategies:

  • Auxin-inducible degron approach for rapid protein depletion

  • Combinatorial depletion to assess compensatory mechanisms

  • Tissue-specific conditional knockouts to evaluate context-dependent functions

• Differential localization analysis:

  • Super-resolution microscopy to precisely map subcellular distributions

  • Live-cell imaging with orthogonally labeled proteins

  • Focus on differences such as INPP5B's enrichment in cis-Golgi/ERGIC versus OCRL1's association with the TGN

• Substrate specificity profiling:

  • In vitro enzyme assays with various phosphoinositide substrates

  • Lipidomic analysis following selective depletion

  • Mass spectrometry to identify specific lipid species affected by each enzyme

This methodological framework will help delineate the unique and overlapping functions of these closely related phosphatases in cellular phosphoinositide metabolism and membrane trafficking.

How can researchers quantitatively assess INPP5B enzymatic activity in different subcellular compartments?

Measuring INPP5B's 5-phosphatase activity in specific subcellular locations requires sophisticated approaches that combine biochemical assays with spatial resolution:

• Fluorescent phosphoinositide biosensors:

  • Express compartment-targeted PH domains that bind PtdIns(4,5)P₂

  • Monitor redistribution following INPP5B activation or inhibition

  • Use FRET-based biosensors for ratiometric quantification

• Organelle isolation and biochemical analysis:

  • Fractionate cells to isolate specific organelles (Golgi, ERGIC, endosomes)

  • Perform lipid extraction and quantitative mass spectrometry

  • Compare phosphoinositide profiles in wild-type vs. INPP5B-depleted samples

• In situ enzyme activity visualization:

  • Use fixed-cell immunohistochemistry with phosphoinositide-specific antibodies

  • Correlate INPP5B localization with phosphoinositide distribution

  • Implement computational image analysis for quantification

• Optogenetic approaches:

  • Develop light-inducible INPP5B recruitment systems

  • Target INPP5B to specific compartments via light stimulation

  • Monitor phosphoinositide depletion and functional consequences

• Single-molecule tracking combined with activity sensors:

  • Track individual INPP5B molecules using super-resolution microscopy

  • Correlate enzyme diffusion with local phosphoinositide turnover

  • Build spatial maps of enzymatic activity

This multi-faceted approach will provide unprecedented insights into the compartment-specific activities of INPP5B and how these contribute to its diverse cellular functions.

What are the most common causes of non-specific binding when using INPP5B antibodies, and how can researchers address them?

Non-specific binding is a significant challenge when working with INPP5B antibodies, particularly due to its structural similarity with OCRL1 and other phosphatases. Researchers can address several common causes:

• Cross-reactivity with related phosphatases:

  • Solution: Validate with knockout controls or peptide competition assays

  • Consider using antibodies targeting unique epitopes (e.g., C-terminal region AA 957-987)

  • Pre-absorb antibodies against recombinant related proteins

• High background in immunohistochemistry:

  • Solution: Optimize blocking conditions (5% BSA or 10% normal serum from same species as secondary antibody)

  • Reduce antibody concentration (optimal range typically 1:100-1:500)

  • Include 0.1-0.3% Triton X-100 for better penetration and reduced background

• Multiple bands in Western blots:

  • Solution: Use gradient gels for better separation

  • Optimize sample preparation (add phosphatase inhibitors to preserve phosphorylation states)

  • Consider alternative epitopes if degradation products are present

• Endogenous peroxidase activity interference (for HRP-conjugates):

  • Solution: Thorough quenching with 0.3% H₂O₂ in methanol for 30 minutes

  • For tissue sections, include sodium azide treatment before antibody application

  • Consider alternative detection methods for highly problematic samples

• Fc receptor binding in immune cells:

  • Solution: Pre-block with unconjugated host IgG or Fc block

  • Use F(ab')₂ or Fab fragments instead of full antibodies

  • Include 1-5% normal serum from the antibody host species

Methodical troubleshooting of these common issues will significantly improve specificity and signal-to-noise ratio when working with INPP5B antibodies.

How can researchers optimize detection sensitivity for low-abundance INPP5B expression?

Detecting low-abundance INPP5B expression requires careful optimization of several experimental parameters:

• Signal amplification strategies:

  • Implement tyramide signal amplification (TSA) for HRP-conjugated antibodies

  • Use polymer-based detection systems rather than standard ABC methods

  • Consider multiple layers of biotinylated reagents for signal enhancement

• Sample preparation optimization:

  • Use antigen retrieval methods appropriate for phosphatases (citrate buffer pH 6.0)

  • Optimize fixation conditions (4% PFA for 10-15 minutes typically preserves epitopes)

  • Include phosphatase inhibitors during all preparation steps

• Antibody concentration and incubation parameters:

  • Extend primary antibody incubation to overnight at 4°C

  • Optimize antibody concentration through titration experiments

  • Consider using antibodies targeting different epitopes in parallel

• Detection system enhancement:

  • Use highly sensitive chemiluminescent substrates for Western blotting

  • Implement longer exposure times with cooled CCD cameras

  • Consider digital accumulation of signal over time

• Pre-enrichment approaches:

  • Implement subcellular fractionation to concentrate target compartments

  • Use immunoprecipitation before Western blotting

  • Consider proximity labeling approaches to identify low-abundance interactors

These optimization strategies will significantly improve detection of low-abundance INPP5B while maintaining specificity and signal-to-noise ratio.

What strategies should researchers employ when results from INPP5B antibody experiments contradict other data?

When facing contradictory results between INPP5B antibody experiments and other data sources, researchers should implement a systematic validation approach:

• Technical validation:

  • Repeat experiments with multiple antibody lots and sources

  • Test antibodies targeting different epitopes (N-terminal, C-terminal, internal domains)

  • Validate with alternative detection methods (fluorescence vs. HRP)

• Biological validation:

  • Implement genetic approaches (siRNA, CRISPR/Cas9 knockout) to confirm specificity

  • Use overexpression systems with tagged INPP5B constructs

  • Implement rescue experiments with wild-type vs. mutant constructs

• Data integration approaches:

  • Correlate protein detection with mRNA expression data

  • Compare with proteomic datasets from similar systems

  • Review literature for similar discrepancies and potential explanations

• Context-dependent expression analysis:

  • Evaluate cell-cycle dependence of INPP5B expression and localization

  • Test multiple cell types to assess tissue-specific differences

  • Examine effects of cellular stress, differentiation, or activation states

• Potential mechanistic explanations:

  • Consider post-translational modifications affecting epitope accessibility

  • Evaluate protein complex formation masking antibody binding sites

  • Assess potential proteolytic processing affecting detection

This systematic approach will help determine whether contradictions arise from technical limitations, biological complexities, or novel regulatory mechanisms affecting INPP5B detection.

How can INPP5B antibodies be used to investigate its role in retrograde ERGIC-to-ER transport?

INPP5B has been implicated in retrograde ERGIC-to-ER transport based on its effect on ERGIC53 distribution . To further investigate this role, researchers can implement several advanced approaches using INPP5B antibodies:

• High-resolution co-localization studies:

  • Employ super-resolution microscopy (STED, STORM, or PALM)

  • Triple-label INPP5B with ERGIC53 and COPI components

  • Quantify co-localization coefficients at tubular-vesicular carriers

• Live trafficking assays:

  • Track ERGIC-to-ER cargo proteins (e.g., KDEL-receptor) in control vs. INPP5B-depleted cells

  • Implement RUSH (retention using selective hooks) system for synchronized cargo release

  • Quantify trafficking kinetics using fluorescence microscopy

• Immunoisolation of transport intermediates:

  • Use magnetic beads coated with INPP5B antibodies to isolate associated membranes

  • Characterize isolated fractions by proteomics and lipidomics

  • Identify novel components of INPP5B-dependent transport pathways

• In vitro reconstitution assays:

  • Prepare semi-intact cells and monitor ERGIC-ER transport with INPP5B antibody inhibition

  • Add back purified INPP5B (wild-type vs. catalytically inactive)

  • Assess role of specific phosphoinositide conversion in transport steps

• Cargo-specific analysis:

  • Examine behavior of model cargo proteins (ERGIC53, KDEL-R, p24 proteins)

  • Assess mislocalization patterns following INPP5B manipulation

  • Correlate phosphoinositide metabolism with cargo transport efficiency

These approaches will provide mechanistic insights into how INPP5B's phosphatase activity and protein interactions regulate retrograde membrane trafficking between the ERGIC and ER.

What experimental approaches can elucidate the functional relationship between INPP5B and B cell receptor signaling for therapeutic applications?

Given INPP5B's recently discovered role in BCR signaling and its potential as a therapeutic target for B cell malignancies , researchers can implement several advanced experimental approaches:

• Patient-derived xenograft (PDX) models:

  • Establish PDX models from B cell malignancy patients

  • Assess correlation between INPP5B expression and disease progression

  • Test effects of INPP5B inhibition on tumor growth and response to standard therapies

• Chemical biology approaches:

  • Develop small molecule inhibitors targeting INPP5B's catalytic domain

  • Implement targeted protein degradation approaches (PROTACs)

  • Assess effects on BCR signaling in malignant vs. normal B cells

• Phosphoproteomic analysis:

  • Compare phosphorylation networks in control vs. INPP5B-depleted B cells

  • Focus on BCR signalosome components

  • Identify novel substrates and signaling nodes affected by INPP5B

• BCR microdomain organization:

  • Use advanced imaging (STORM, PALM) to visualize BCR nanocluster organization

  • Correlate with phosphoinositide distribution using specific probes

  • Track reorganization dynamics following antigen engagement

• Combination therapy testing:

  • Assess synergy between INPP5B inhibition and established BCR pathway inhibitors (BTK, SYK)

  • Implement high-throughput drug screens to identify synthetic lethal interactions

  • Develop rational combination strategies based on pathway crosstalk

These approaches will help translate basic findings about INPP5B's role in BCR signaling into potential therapeutic applications for B cell malignancies, particularly those dependent on chronic active BCR signaling.