OCRL Antibody

What is the most reliable method to detect OCRL protein expression in cell and tissue samples?

Western blotting remains the gold standard for detecting OCRL protein expression. For optimal results:

• Use 40-50 μg of total protein from cell or tissue lysates

• Run samples on 7.5-10% gels for optimal resolution of the 105 kDa OCRL protein

• Transfer to nitrocellulose membrane using standard protocols

• Block with 5% non-fat dried milk in PBS

• Incubate with anti-OCRL antibody at 1:500-1:2000 dilution

• Detect with appropriate secondary antibodies and visualization systems

OCRL consistently appears as a 105 kDa band in Western blots, aligning with its calculated molecular weight of 103 kDa . When experimenting with new cell lines or tissues, include positive controls such as HeLa cells, HEK-293 cells, or brain tissue (human, mouse, or rat), which are known to express detectable levels of OCRL .

Which antibody applications are validated for OCRL research, and what are their optimal conditions?

OCRL antibodies have been validated for multiple applications, each requiring specific optimization:

For IHC applications, OCRL antibodies have been successfully used on kidney tissue sections, where they reveal specific staining patterns in tubular structures . For IF applications, OCRL typically shows Golgi-predominant localization in resting cells, with additional distribution to endosomes and plasma membrane .

How can researchers validate the specificity of OCRL antibodies?

Rigorous validation of OCRL antibodies is essential for reliable experimental outcomes:

• Genetic validation:

  • Compare staining in normal versus Lowe syndrome patient fibroblasts (which lack OCRL)

  • Use OCRL knockdown/knockout cells as negative controls

  • Perform rescue experiments with wildtype OCRL re-expression

• Biochemical validation:

  • Western blot should show a single predominant band at ~105 kDa

  • Peptide competition assays can confirm epitope specificity

  • Immunoprecipitation followed by mass spectrometry can verify pulled-down protein identity

• Cross-reactivity assessment:

  • Test across multiple species if cross-species reactivity is claimed

  • Evaluate in tissues known to express OCRL versus those with minimal expression

Research has demonstrated OCRL antibody specificity by showing a single 105 kDa protein in normal fibroblasts that is absent in fibroblasts from OCRL patients who lack OCRL transcript . Additionally, co-staining with markers for specific cellular compartments should show the expected localization pattern, primarily at the Golgi complex in unstimulated cells .

How can OCRL antibodies be utilized to study dynamic changes in OCRL localization during cell signaling?

OCRL undergoes stimulus-dependent translocation between cellular compartments, which can be effectively studied using antibodies:

• Capturing translocation events:

  • Fix cells at different time points after stimulation

  • Use OCRL antibodies alongside organelle markers

  • Quantify colocalization coefficients to measure redistribution

• Complementary approaches:

  • Perform subcellular fractionation followed by Western blotting with OCRL antibodies

  • Compare with live-cell imaging of fluorescently tagged OCRL

  • Correlate localization changes with functional outcomes

Research has demonstrated that anti-CD3 stimulation induces OCRL translocation from the Golgi to the plasma membrane in T-cells, with corresponding changes in the distribution of OCRL between subcellular fractions . This translocation is ORP4L-dependent and correlates with changes in PI(4,5)P₂ levels at the plasma membrane .

For quantification, researchers should:

• Measure the percentage of OCRL colocalization with compartment markers

• Analyze at least two sections per cell, ensuring peripheral and perinuclear structures are equally represented

• Compare signal intensities across cellular compartments before and after stimulation

What methodological approaches can researchers use to study OCRL's role in clathrin-mediated endocytosis?

OCRL plays a critical role in clathrin-mediated endocytosis, which can be examined using multiple antibody-based approaches:

• Characterizing protein interactions:

  • Use OCRL antibodies for co-immunoprecipitation to pull down clathrin, AP-2, and other endocytic proteins

  • Perform reciprocal IPs using antibodies against endocytic proteins to pull down OCRL

  • Compare wildtype versus mutant OCRL interactions

• Localization studies:

  • Co-stain for OCRL alongside clathrin, AP-2, and SNX9

  • Analyze distribution patterns in normal versus Lowe syndrome cells

  • Quantify clustering of endocytic structures

• Functional assays:

  • Examine receptor internalization and recycling in OCRL-depleted cells

  • Measure PI(4,5)P₂ levels using specific probes

  • Rescue experiments with wildtype versus mutant OCRL

Research has shown that Lowe syndrome patient fibroblasts lacking OCRL display increased punctate immunoreactivity for clathrin, AP-2, and particularly SNX9, indicating accumulated endocytic structures . This phenotype correlates with defective endocytosis and can be rescued by reintroduction of wildtype OCRL .

How should researchers interpret OCRL antibody signals in primary cilia studies?

OCRL localization to primary cilia represents an important research area, requiring careful methodological considerations:

• Sample preparation:

  • Use serum starvation to induce ciliation in cultured cells

  • Apply gentle fixation protocols to preserve ciliary structures

  • Co-stain with ciliary markers like acetylated α-tubulin

• Detection strategies:

  • Employ super-resolution microscopy for precise localization

  • Use confocal z-stacks to capture the entire cilium

  • Apply deconvolution for improved signal resolution

• Validation in tissues:

  • Examine OCRL localization in tissues with prominent cilia

  • Kidney tubular cells and retinal photoreceptors are particularly valuable models

Research has demonstrated OCRL localization to the primary cilium in retinal pigment epithelial cells, fibroblasts, and kidney tubular cells . In retinal tissue, OCRL localizes to photoreceptor outer segments, which are extensions of specialized photoreceptor sensory cilia . This localization pattern provides important insights into potential mechanisms underlying the ocular and renal manifestations of Lowe syndrome.

What are the most common technical challenges when using OCRL antibodies for immunoprecipitation studies?

Immunoprecipitation (IP) with OCRL antibodies presents several challenges that require methodological adjustments:

• Optimizing extraction conditions:

  • OCRL associates with membranes and cytoskeletal elements, requiring appropriate lysis buffers

  • Mild detergents (0.5-1% NP-40 or Triton X-100) preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain phosphorylation states

• Antibody selection and usage:

  • Use 0.5-4.0 μg antibody per 1-3 mg of total protein lysate

  • Consider covalently coupling antibodies to beads to prevent IgG contamination

  • Pre-clear lysates to reduce non-specific binding

• Detecting interaction partners:

  • OCRL interacts with numerous proteins including clathrin, AP-2, and SNX9

  • Use appropriate controls to distinguish specific from non-specific interactions

  • Compare wildtype versus mutant OCRL to identify domain-specific interactions

Research has successfully used OCRL antibodies to capture protein complexes involved in membrane trafficking. For example, immunoprecipitation studies have revealed that mutations in OCRL's clathrin-binding domains disrupt interactions with CI-M6PR, EpsinR, and PI3KcIIα, while interaction with SNX9 persists through different binding sites .

How can researchers address variable results when using OCRL antibodies across different cell types or tissues?

Variability in OCRL antibody performance across experimental systems requires systematic troubleshooting:

• Expression level differences:

  • OCRL expression varies naturally between tissues and cell types

  • Adjust antibody concentrations proportionally to expected expression levels

  • Include positive controls with known OCRL expression

• Epitope accessibility issues:

  • OCRL's conformation or interaction partners may mask epitopes in certain contexts

  • Test multiple antibodies targeting different OCRL regions

  • Optimize fixation and permeabilization protocols for each cell type

• Subcellular distribution variations:

  • OCRL localization patterns may differ between cell types

  • Co-stain with compartment markers appropriate for each cell type

  • Quantify relative distribution across cellular compartments

OCRL antibodies have been successfully used across human, mouse, and rat samples, with reliable detection in various cell types including HeLa, HEK-293, SH-SY5Y, and primary fibroblasts . For tissue analysis, OCRL antibodies work well in brain, kidney, and retinal tissues, though optimization may be required for each specific tissue type .

What controls and experimental design approaches are essential when studying disease-associated OCRL mutations?

When investigating disease-associated OCRL mutations, rigorous experimental design is crucial:

• Control selection:

  • Include both wildtype OCRL and known pathogenic mutants

  • Use patient-derived cells when available

  • Include rescue experiments with various OCRL constructs

• Functional domain analysis:

  • Compare mutations affecting different OCRL domains:

    • 5-phosphatase domain (e.g., V527D) for catalytic activity

    • Clathrin-binding motifs for endocytic functions

    • Protein interaction domains for complex formation

• Readout parameters:

  • Measure multiple outcomes including:

    • PI(4,5)P₂ levels using specific biosensors

    • Protein localization across cellular compartments

    • Endocytic trafficking efficiency

    • Protein-protein interactions

Research has demonstrated that wildtype OCRL re-expression can rescue PI(4,5)P₂ accumulation in OCRL-depleted cells, whereas expression of mutant OCRL incapable of binding interaction partners fails to rescue this phenotype . This approach distinguishes between mutations affecting catalytic activity versus protein-protein interactions.

How can OCRL antibodies contribute to studying the role of OCRL in immune cell function?

Recent research has revealed important functions for OCRL in immune cells, particularly T-cells:

• T-cell receptor signaling:

  • OCRL translocates from Golgi to plasma membrane upon TCR stimulation

  • This translocation is dependent on ORP4L interaction

  • Antibodies can track this dynamic process through fixed-time-point analysis

• Methodological approaches:

  • Immunofluorescence with OCRL antibodies before and after T-cell stimulation

  • Subcellular fractionation followed by Western blotting

  • Co-immunoprecipitation to identify stimulus-dependent interaction partners

• Functional correlates:

  • Link OCRL localization to PI(4,5)P₂ metabolism

  • Correlate with downstream signaling events

  • Connect to T-cell activation outcomes

Research has shown that anti-CD3 stimulation induces OCRL translocation from the Golgi to the plasma membrane in Jurkat T-cells, increasing colocalization with ORP4L . This translocation is functionally important, as it regulates PI(4,5)P₂ levels at the plasma membrane, which in turn affects T-cell receptor signaling .

What are the key considerations when using OCRL antibodies alongside phosphoinositide biosensors?

Combining OCRL antibody staining with phosphoinositide biosensors requires careful methodological planning:

• Fixation compatibility:

  • Choose fixation protocols that preserve both protein epitopes and lipid distribution

  • PFA fixation (4%, 10-15 minutes) generally works well for both

  • Avoid methanol fixation which can extract membrane lipids

• Detection strategies:

  • For fixed samples, combine OCRL immunostaining with lipid-binding domain probes

  • Use the PH domain of PLCδ1 as a PI(4,5)P₂ sensor

  • Consider sequential staining protocols to minimize interference

• Validation approaches:

  • Compare OCRL wildtype versus phosphatase-dead mutants

  • Analyze cells before and after stimulation that triggers PI(4,5)P₂ hydrolysis

  • Include OCRL knockout/knockdown cells as controls

Research has successfully employed PI(4,5)P₂ indicators such as the GFP-PH PLCδ1 domain alongside OCRL antibodies or OCRL constructs . This combination has revealed that OCRL knockdown results in PI(4,5)P₂ accumulation at the plasma membrane, while anti-CD3 stimulation decreases PI(4,5)P₂ levels in control cells but not in OCRL knockdown cells .

How should researchers interpret data from experiments combining OCRL antibodies with proximity labeling approaches?

Proximity labeling techniques offer powerful insights when combined with traditional antibody approaches:

• Complementary strengths:

  • Proximity labeling (BioID, APEX) identifies neighbors in live cells

  • Antibodies confirm specific interactions and localizations

  • Together they provide both discovery and validation

• Experimental design:

  • Use OCRL antibodies to validate proximity labeling hits

  • Compare interactomes across different cellular compartments

  • Analyze changes in interaction networks upon stimulation

• Data interpretation:

  • Distinguish between direct binding partners and proteins in the same complex

  • Consider dynamic versus stable interactions

  • Evaluate functional relevance through knockout/knockdown approaches

The OCRL interactome includes numerous proteins involved in membrane trafficking, with particularly strong representation of proteins involved in clathrin-dependent transport . Antibody-based validation of these interactions has confirmed binding partners including clathrin, AP-2, Rab proteins, and SNX9 .

How can researchers reconcile differences in results obtained using different OCRL antibodies?

When different OCRL antibodies yield inconsistent results, systematic analysis is essential:

• Antibody characterization:

  • Compare epitope locations on the OCRL protein

  • Evaluate validation data for each antibody

  • Test antibodies side-by-side under identical conditions

• Biological explanations:

  • Different epitopes may be differentially accessible in various cellular contexts

  • Post-translational modifications might affect antibody binding

  • Protein conformational changes could expose or mask epitopes

• Resolution strategies:

  • Use multiple detection methods to cross-validate findings

  • Employ genetic approaches (tagged OCRL constructs, CRISPR editing)

  • Consider using antibodies against different OCRL epitopes in combination

For example, if one antibody shows predominantly Golgi localization while another detects more endosomal staining, this might reflect either technical differences in epitope accessibility or biological differences in OCRL conformation at different locations.

What methodological approaches allow integration of OCRL antibody data with insights from other experimental systems?

Comprehensive OCRL research requires integration across multiple methodological approaches:

• Correlative microscopy:

  • Combine immunofluorescence with electron microscopy

  • Use OCRL antibodies for immuno-EM to achieve ultrastructural localization

  • Correlate light and electron microscopy data

• Functional genomics integration:

  • Connect antibody-based observations with CRISPR/siRNA screens

  • Validate genetic hits using antibody-based approaches

  • Combine protein-level and transcript-level analyses

• Disease model correlation:

  • Compare antibody findings between patient-derived cells and model systems

  • Validate animal model observations in human samples

  • Connect cellular phenotypes to clinical manifestations

Research has successfully combined multiple approaches, including immunofluorescence, biochemical fractionation, and electron microscopy to characterize OCRL's roles in endocytic trafficking . For example, immunoelectron microscopy using anti-OCRL antibodies has helped define its precise localization within endocytic structures .