INPP5E Antibody

What is INPP5E and why is it significant in research?

INPP5E (inositol polyphosphate-5-phosphatase, 72 kDa) is a critical enzyme involved in phosphoinositide metabolism that regulates various cellular processes. Recent studies have identified it as a key player in the phosphoinositide manipulation at immune synapses, controlling TCR signaling cascades . INPP5E has a calculated molecular weight of 644 amino acids and approximately 70 kDa, though it is often observed at 64-66 kDa in experimental conditions . Its importance extends to primary cilia function, with mutations being associated with ciliopathies.

What types of INPP5E antibodies are available for research purposes?

Current research applications utilize several antibody formats targeting INPP5E:

• Polyclonal antibodies (e.g., rabbit polyclonal IgG) that recognize multiple epitopes

• Monoclonal antibodies (e.g., mouse IgG1) that provide highly specific single-epitope binding

• Antibodies targeting different regions of INPP5E, including:

  • Full-length protein (AA 1-644)

  • N-terminal regions (AA 1-133)

  • C-terminal regions

  • Specific internal domains (AA 510-630)

How should I determine the optimal INPP5E antibody for my specific research application?

Selection should be based on:

What are the validated tissue and cell models for INPP5E antibody applications?

INPP5E antibodies have been validated in diverse biological samples, with specific positive results in:

How should I design proper controls for INPP5E antibody experiments?

For rigorous experimental design, implement the following controls:

• Positive controls: Utilize tissues/cells with known INPP5E expression (e.g., HEK-293 cells, mouse brain tissue)

• Negative controls:

  • Primary antibody omission

  • INPP5E knockdown validation through siRNA as demonstrated in recent studies where Western blot confirmed depletion of the ~70 kDa INPP5E signal

• Specificity controls: Secondary antibody-only controls to detect non-specific binding, which has been observed in both T cells and APCs in immune synapse studies

What are the recommended storage and handling protocols for INPP5E antibodies?

Different INPP5E antibody formulations require specific storage conditions:

• Standard formulations: Store at -20°C with PBS containing 0.02% sodium azide and 50% glycerol (pH 7.3). Stable for one year after shipment

• Conjugation-ready formulations: Store at -80°C in PBS-only buffer (BSA and azide-free) at 1 mg/mL concentration

• For standard formulations, aliquoting is unnecessary for -20°C storage

• Some specialized formulations (20μl sizes) contain 0.1% BSA

How can I effectively track INPP5E localization during immune synapse formation?

Recent research has revealed dynamic INPP5E distribution during immune synapse formation:

• INPP5E signals appear faintly at the immune synapse 2 minutes after T cell-APC conjugation

• Signal intensity increases 5-10 minutes after conjugation

• In resting cells, INPP5E localizes to centrioles, but relocates toward the immune synapse upon stimulation

• For optimal visualization, immunostaining combined with 3D-structured illumination microscopy (3D-SIM) provides detailed spatial resolution

What methodological approaches can validate INPP5E knockdown efficiency?

Effective validation requires multi-parameter assessment:

• Western blot: Confirm depletion of INPP5E signal at approximately 70 kDa compared to control cells

• Immunofluorescence: Assess reduction in INPP5E enrichment at centrioles in resting cells

• Functional assays: Measure decreased INPP5E polarization toward the immune synapse (e.g., reduced from 71.52% in control cells to 38.09% in knockdown cells)

• Quantification: Express results as percentage of conjugation events showing proper localization

How should I approach colocalization studies involving INPP5E?

For accurate colocalization analysis:

• Employ triple-staining techniques with INPP5E antibody and known cellular markers

• Utilize super-resolution microscopy (e.g., 3D-SIM) for precise spatial mapping

• For centriole/basal body localization, combine with centrosomal markers

• For immune synapse studies, measure polarization toward the T cell-APC interface

• Quantify colocalization using appropriate software and statistical metrics (Pearson's correlation, Manders' overlap coefficient)

Why might I observe discrepancies between calculated and observed molecular weights for INPP5E?

While the calculated molecular weight of INPP5E is 70 kDa (644 amino acids), Western blot typically detects it at 64-66 kDa . This discrepancy may result from:

• Post-translational modifications affecting protein migration

• Proteolytic processing in specific cell types

• Secondary/tertiary protein structure influencing SDS-PAGE mobility

• Alternative splicing generating different isoforms

The consistent observation across multiple studies suggests this is a normal characteristic rather than an experimental artifact.

How can I optimize signal-to-noise ratio when using INPP5E antibodies in immunofluorescence?

Non-specific binding has been reported in both T cells and APCs . To improve signal specificity:

• Titrate antibody concentration (recommended range: 1:20-1:200)

• Extend blocking time with 5% BSA or serum matching secondary antibody host

• Include 0.1-0.3% Triton X-100 for improved penetration

• Consider antigen retrieval optimization (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Employ confocal microscopy with appropriate negative controls

• Use fluorophore-conjugated secondary antibodies with minimal spectral overlap

What are critical considerations when interpreting INPP5E localization in dynamic cellular processes?

When analyzing INPP5E distribution during processes like immune synapse formation:

• Account for temporal dynamics (signals increase 2-10 minutes after cell conjugation)

• Consider multiple z-stacks to capture the complete 3D distribution

• Recognize that antibody accessibility may vary between cellular compartments

• Compare localization patterns with known INPP5E interaction partners

• Analyze multiple time points to establish the sequence of recruitment events

• Quantify signal intensity changes across experimental conditions

How does INPP5E contribute to immune synapse function and TCR signaling?

Recent discoveries highlight INPP5E as a novel regulator of T cell function:

• INPP5E regulates CD3ζ enrichment at the immune synapse

• It participates in phosphoinositide manipulation at the synapse, controlling TCR signaling cascades

• Knockdown experiments demonstrate its critical role in immune synapse formation events

• The spatial-temporal distribution of INPP5E during immune synapse formation follows a specific pattern, appearing 2 minutes after conjugation and increasing 5-10 minutes after conjugation

What emerging methods are advancing INPP5E functional studies?

Cutting-edge approaches include:

• Super-resolution microscopy, particularly 3D-structured illumination microscopy (3D-SIM), for detailed visualization of INPP5E localization

• Matched antibody pairs developed specifically for cytometric bead arrays and multiplex assays

• Conjugation-ready antibody formats allowing flexible application in ELISAs, multiplex assays, mass cytometry, and multiplex imaging

• Combined knockdown and live cell imaging approaches to track dynamic INPP5E redistribution

How can researchers integrate INPP5E studies into broader signaling pathway analyses?

For comprehensive signaling studies:

• Combine INPP5E antibodies with phosphoinositide sensors to correlate enzyme localization with substrate dynamics

• Utilize matched antibody pairs for multiplexed detection in complex samples

• Integrate INPP5E research with investigation of interacting partners identified through co-immunoprecipitation studies

• Apply systems biology approaches to position INPP5E within larger phosphoinositide metabolism networks

• Correlate INPP5E activity with downstream signaling events such as TCR cascade activation