RPGR Antibody

What is RPGR and why is it significant in vision research?

RPGR (retinitis pigmentosa GTPase regulator) is a protein with a series of six RCC1-like domains (RLDs), characteristic of highly conserved guanine nucleotide exchange factors. It plays a crucial role in ciliogenesis, likely by regulating actin stress filaments and cell contractility. RPGR is essential for photoreceptor integrity and is involved in microtubule organization and regulation of transport in primary cilia . The gene undergoes complex alternative splicing, encoding multiple protein isoforms that perform overlapping yet somewhat distinct transport-related functions in photoreceptors . Mutations in the RPGR gene are a frequent cause of retinal degeneration, particularly X-linked retinitis pigmentosa (XLRP), making it a significant target for vision research .

What are the major RPGR isoforms and how do they differ?

RPGR has two primary isoforms: RPGR 1-19 and RPGR ORF15. The RPGR 1-19 isoform (expected molecular mass ~90 kDa) contains exons 1-19, while the RPGR ORF15 isoform (expected molecular mass ~140 kDa) contains an alternative terminal exon (ORF15) that replaces exons 16-19 . Western blot analyses have revealed multiple bands for both isoforms:

The higher molecular weight bands and variations in detected sizes may represent post-translationally modified isotypes or alternatively spliced isoforms .

How should I select the appropriate RPGR antibody for my specific experimental needs?

Selection of an appropriate RPGR antibody depends on:

• Target epitope: Choose antibodies targeting different regions depending on which isoform you want to detect:

  • For RPGR 1-19: Use antibodies targeting exon 19 (e.g., RPGR-E19 antibody)

  • For RPGR ORF15: Use antibodies targeting the ORF15 C-terminus (e.g., ORF15 CP, CT-15, ORF15 C2)

  • For both isoforms: Use antibodies targeting common regions (e.g., GR-P1 targeting exon 1)

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF/ICC)

• Species reactivity: Ensure the antibody reacts with your species of interest. Note that RPGR expression levels are lower in mouse compared to human and bovine retinal extracts

• Subcellular localization: Select antibodies validated for the cellular compartment you're investigating. Some antibodies may perform better for connecting cilia versus outer segment localization

What are the optimal conditions for using RPGR antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) with RPGR antibodies:

• Fixation and tissue processing: Use formalin/PFA-fixed paraffin-embedded sections

• Antigen retrieval:

  • Heat-induced epitope retrieval at pH 6.0 is recommended for most RPGR antibodies

  • Some antibodies may require TE buffer pH 9.0 as an alternative retrieval method

• Dilution ranges:

  • Use dilutions of 1:50 to 1:500 depending on the specific antibody

  • Novus Biologicals antibody: 1:50 dilution

  • Abcam antibodies: 1:50 dilution

  • Proteintech antibody: 1:50-1:500 dilution

• Tissue recommendations: RPGR antibodies have been successfully used on:

  • Human tissues: testis, fallopian tube, heart, kidney

  • Mouse tissues: eye, kidney

• Signal detection: Use appropriate secondary antibodies and visualization systems based on the host species of your primary antibody (typically rabbit or mouse)

How do I interpret complex banding patterns in Western blots using RPGR antibodies?

When interpreting Western blots with RPGR antibodies:

• Expected molecular weights:

  • The calculated molecular weight of RPGR is 113.4 kDa

  • The expected apparent molecular weight of full-length RPGR ORF15 is ~140 kDa, but it may migrate at an aberrant rate due to its highly acidic carboxyl-terminal region

  • RPGR 1-19 has an expected molecular mass of ~90 kDa

  • Observed molecular weights from Proteintech antibody: 100-105 kDa and 70 kDa

• Multiple bands interpretation:

  • Multiple bands may represent different isoforms, post-translational modifications, or alternatively spliced variants

  • Lower molecular weight bands (100-120 kDa) for RPGR ORF15 may represent proteolytically processed fragments

  • Higher molecular weight bands (240-250 kDa) for RPGR ORF15 may represent post-translational modifications

  • An 80 kDa band seen with wild-type RPGR in some experiments may represent an aberrantly spliced RPGR variant with a large C-terminal deletion

• Confirmation strategies:

  • Use multiple antibodies targeting different epitopes to confirm isoform identity

  • Perform peptide competition assays to verify specificity (pre-incubation with specific peptide should eliminate the immunoreactive signal)

  • Include positive controls from tissues known to express RPGR (retina, eye tissue)

What controls should be included when using RPGR antibodies in experimental settings?

Essential controls for RPGR antibody experiments include:

• Positive tissue controls:

  • Retina/eye tissue (highest expression)

  • Additional validated tissues: testis, fallopian tube, heart, kidney

• Negative controls:

  • Pre-immune serum

  • Secondary antibody only (omitting primary antibody)

  • Peptide competition assays (pre-incubation with specific peptide)

• Recombinant protein controls:

  • Xpress-tagged Rpgr exon 16-19-derived protein in transfected cells

  • RPGR fusion proteins from expression vectors

• Cellular controls for localization studies:

  • hTERT-RPE1 cells (positive for IF/ICC)

  • Y79 cells, SH-SY5Y cells (positive for WB)

• Species-specific considerations:

  • Mouse samples show less expression compared to human and bovine samples

  • Include species-appropriate positive controls based on antibody reactivity

How can RPGR antibodies be used to investigate post-translational modifications of the protein?

RPGR antibodies can be powerful tools for studying post-translational modifications, particularly glutamylation:

• Glutamylation detection methodology:

  • Use antibodies specific for glutamylation (e.g., GT335) in parallel with RPGR antibodies on the same samples

  • Perform co-immunoprecipitation followed by Western blotting with both antibody types

  • Look for immunoreactive bands of the same molecular weight in both blots to confirm glutamylated RPGR

• Data interpretation:

  • RPGR expressed from both wild-type and codon-optimized constructs shows similar glutamylation patterns in vitro

  • Glutamylation is typically detected on full-length RPGR protein but not on truncated variants

  • Non-glutamylated bands may represent aberrantly spliced variants with C-terminal deletions

• Experimental approach for comparing wild-type and modified RPGR:

  • Express RPGR variants in HEK293T cells

  • Perform Western blotting with RPGR antibodies

  • Perform parallel Western blotting with GT335 antibody

  • Compare banding patterns to identify which RPGR variants are glutamylated

This approach has been particularly valuable in gene therapy research to confirm that codon-optimized RPGR sequences produce proteins with post-translational modifications similar to wild-type RPGR .

Why do studies report discrepancies in RPGR localization between species and how can antibodies help resolve this?

Discrepancies in RPGR localization between species represent an important research question that can be addressed with carefully selected antibodies:

• Observed localization differences:

  • RPGR is consistently localized to the photoreceptor connecting cilia (CC) across species

  • Some studies report RPGR in the outer segment (OS) in humans and amphibians but not in rodents or pigs

  • Contradicting reports exist regarding bovine OS localization

• Factors contributing to discrepancies:

  • Different antibodies targeting different epitopes or isoforms

  • Variation in tissue processing procedures

  • Species differences in OS structure (multiple superficial incisures in humans/amphibia vs. single deep incisures in rodent/bovine/canine OS)

• Methodological approach to resolve discrepancies:

  • Use multiple antibodies targeting different RPGR domains on the same tissue

  • Apply consistent tissue processing across species

  • Include proper controls for each antibody

  • Document the specific antibody epitope, processing method, and detection system

  • Compare localization in dividing cells (centrosomes) vs. non-dividing cells (transition zone of ciliary axoneme)

This careful approach can help determine whether localization differences represent true biological variation or technical artifacts.

How can RPGR antibodies be utilized in gene therapy and CRISPR/Cas9 research?

RPGR antibodies play critical roles in advancing gene therapy and CRISPR/Cas9 research:

• Gene augmentation therapy validation:

  • Use isoform-specific antibodies to confirm expression of the correct RPGR protein from AAV vectors

  • Compare glutamylation patterns between wild-type and therapeutically delivered RPGR to ensure proper post-translational modification

  • Monitor stability and expression levels of codon-optimized vs. wild-type RPGR sequences

• CRISPR/Cas9 gene editing assessment:

  • Use domain-specific antibodies to demonstrate restoration of full-length RPGR ORF15 protein after editing

  • Analyze protein expression in treated vs. untreated retinas to confirm successful correction

  • Employ antibodies against RPGR-interacting proteins to verify restoration of proper molecular interactions

• Experimental design considerations:

  • Select antibodies that can distinguish between endogenous and therapeutically delivered RPGR

  • Include controls for non-specific bands in Western blots, particularly when analyzing complex tissue lysates

  • Use antibodies targeting different domains to confirm full-length protein expression

  • Combine antibody-based detection with functional assays to confirm therapeutic efficacy

These approaches have been successfully implemented to demonstrate that both codon-optimized RPGR gene augmentation and CRISPR/Cas9-mediated excision of mutations can restore RPGR expression in vivo .

What are common challenges in using RPGR antibodies and how can they be addressed?

Common challenges with RPGR antibodies include:

• Multiple bands and interpretation difficulties:

  • Solution: Use multiple antibodies targeting different epitopes to confirm specificity

  • Perform peptide competition assays to identify specific vs. non-specific bands

  • Include purified recombinant RPGR as a positive control

• Inconsistent detection of specific isoforms:

  • Solution: The RPGR ORF15-3 isoform (~140 kDa) is not consistently detected, which may indicate it is unstable, expressed at very low levels, or post-translationally modified

  • Similarly, RPGR 1-19-2 (~110 kDa) is not consistently detectable in whole retinal homogenates but is enriched in cytosolic fractions

  • Use subcellular fractionation to enrich for specific isoforms

• Species-specific expression levels:

  • Solution: Mouse retina shows lower RPGR expression compared to human and bovine samples

  • Adjust loading amounts or exposure times accordingly when comparing across species

  • Use species-matched positive controls

• Antibody cross-reactivity:

  • Solution: Verify antibody specificity using knockout/knockdown models when available

  • Test multiple antibodies targeting different epitopes

  • Perform careful titration to determine optimal antibody concentration

How can I validate the specificity of RPGR antibodies?

To validate RPGR antibody specificity:

• Expression system validation:

  • Test antibody against recombinant RPGR expressed in transfected cells

  • The RPGR-E19 antibody should recognize Xpress-tagged Rpgr exon 16-19-derived protein in transiently-transfected COS-7 cells

  • Compare immunoreactivity patterns between transfected and non-transfected cells

• Peptide competition assays:

  • Pre-incubate antibody with specific peptide versus non-specific peptide

  • Specific peptide should eliminate the immunoreactive signal for antibodies like ORF15 CP in immunoblot analyses

• Multiple antibody approach:

  • Use antibodies targeting different epitopes (e.g., ORF15 CP and RPGR-E19) to differentiate between RPGR isoforms

  • Consistent detection with multiple antibodies increases confidence in specificity

• Western blotting controls:

  • Include positive controls from tissues known to express RPGR (retina, eye tissue)

  • Use pre-immune serum as a negative control

  • Look for expected molecular weight bands (considering post-translational modifications)

• Immunohistochemistry validation:

  • Verify cellular localization is consistent with known RPGR distribution (connecting cilia in photoreceptors)

  • Include appropriate tissue processing controls

What factors affect the reproducibility of experiments using RPGR antibodies?

Key factors affecting reproducibility include:

• Antibody storage and handling:

  • Store at recommended temperature (typically -20°C)

  • Avoid repeated freeze-thaw cycles

  • For some antibodies, aliquoting is unnecessary for -20°C storage

• Sample preparation variables:

  • Tissue fixation method and duration

  • Antigen retrieval conditions (pH and temperature)

  • Protein extraction methods for Western blotting

  • Buffer composition (some antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol pH 7.3)

• Technical considerations:

  • Antibody dilution (optimize for each application: WB: 1:500-1:1000, IHC: 1:50-1:500, IF/ICC: 1:10-1:100)

  • Incubation time and temperature

  • Detection system sensitivity

  • Blocking reagents and washing protocols

• Biological variables:

  • Species differences in RPGR expression levels (mouse < human/bovine)

  • Tissue-specific expression patterns

  • Disease state or experimental conditions affecting RPGR expression

• Reporting standards:

  • Document the specific antibody used (including catalog number and lot)

  • Report all experimental conditions in detail

  • Include all controls used to validate specificity

Adherence to these considerations can significantly improve the reproducibility of experiments using RPGR antibodies across different laboratories and research contexts.

How might new RPGR antibodies advance our understanding of RPGR-related diseases?

Development of new RPGR antibodies could advance the field through:

• Improved isoform specificity:

  • Antibodies targeting unique splice junctions could better differentiate between closely related isoforms

  • Antibodies specific to novel alternatively spliced variants may uncover previously unrecognized RPGR functions

• Post-translational modification detection:

  • Modification-specific antibodies (beyond glutamylation) could illuminate regulatory mechanisms

  • Phosphorylation-specific antibodies might reveal signaling pathways controlling RPGR function

• Species-optimized reagents:

  • Development of antibodies with improved cross-species reactivity would facilitate comparative studies

  • Species-specific antibodies could help resolve contradictory localization findings

• Application-optimized formats:

  • Super-resolution microscopy-compatible antibodies could provide higher-resolution localization data

  • Antibody pairs validated for proximity ligation assays could better characterize protein-protein interactions

• Disease-relevant epitopes:

  • Antibodies recognizing disease-associated mutant forms could facilitate personalized medicine approaches

  • Conformation-specific antibodies might distinguish between functional and non-functional RPGR states

These advances would contribute to better understanding RPGR biology and developing more targeted therapeutic approaches.

What role do RPGR antibodies play in evaluating emerging gene therapies?

RPGR antibodies are critical tools for evaluating gene therapy approaches:

• Expression verification:

  • Confirm expression of the correct RPGR protein from gene therapy vectors

  • Verify that codon-optimized sequences produce proteins with appropriate post-translational modifications

  • Ensure stability and expression levels of therapeutically delivered RPGR

• Localization assessment:

  • Confirm proper subcellular localization of therapeutically expressed RPGR to connecting cilia

  • Verify restoration of normal protein trafficking in treated photoreceptors

• Functional evaluation:

  • Assess restoration of RPGR-protein interactions through co-immunoprecipitation

  • Monitor downstream effects on interacting proteins and cellular structures

• Therapeutic efficacy markers:

  • Use changes in RPGR localization or expression as biomarkers for treatment response

  • Quantify restoration of normal RPGR patterns in treated versus untreated retinas

• Safety monitoring:

  • Detect potential overexpression or mislocalization of therapeutic RPGR

  • Identify unexpected RPGR variants that might arise from gene therapy vectors

These applications make RPGR antibodies essential tools for the continued development of gene therapies for XLRP and related disorders.

How can RPGR antibodies contribute to understanding ciliopathies beyond retinal degeneration?

RPGR antibodies can expand our understanding of ciliopathies through:

• Comparative tissue analysis:

  • Examine RPGR expression and localization in multiple ciliated tissues

  • Compare photoreceptor connecting cilia with primary cilia in other cell types

  • Investigate RPGR in tissues affected in syndromic ciliopathies

• Developmental studies:

  • Track RPGR expression during ciliogenesis and tissue development

  • Examine potential roles in centrosomes of dividing cells versus transition zones in non-dividing cells

• Protein interaction network mapping:

  • Use RPGR antibodies for co-immunoprecipitation to identify tissue-specific interaction partners

  • Compare RPGR complexes across different ciliated tissues

  • Investigate how RPGR mutations affect these interaction networks

• Functional conservation analysis:

  • Compare RPGR localization and function across evolutionarily diverse species

  • Determine which aspects of RPGR function are conserved in different ciliary contexts

• Disease mechanism investigation:

  • Examine RPGR in animal models of non-retinal ciliopathies

  • Investigate potential roles in spermatogenesis and intraflagellar transport processes