GLIPR1L1 Antibody

What is the molecular weight discrepancy observed with GLIPR1L1 in Western blotting?

GLIPR1L1 (GLI pathogenesis-related 1 like 1) has a calculated molecular weight of approximately 27 kDa, but researchers frequently observe bands at different molecular weights. Commercial antibodies report observed molecular weights ranging from 24-68 kDa . This discrepancy is due to:

• Post-translational modifications

• Multiple isoforms (at least two known isoforms)

• Formation of protein complexes

Research methodology recommendation: When performing Western blot analysis, prepare samples under both reducing and native conditions to validate isoform detection. Under reducing conditions, GLIPR1L1 antibodies typically detect a predominant band at 37 kDa, while native conditions reveal high molecular weight complexes (200-1000 kDa) .

Which tissue samples are appropriate positive controls for GLIPR1L1 antibody validation?

Based on multiple antibody validation studies, the following tissues consistently show positive GLIPR1L1 expression:

For cellular models, PC-3 cells have been validated for flow cytometry applications with GLIPR1L1 antibodies . When validating a new GLIPR1L1 antibody, testis tissue lysates provide the most reliable positive control across species.

How can I validate that my GLIPR1L1 antibody detects native protein complexes in sperm biology research?

GLIPR1L1 forms multimeric complexes with other proteins, particularly IZUMO1, which are critical for sperm-egg binding. To validate complex detection:

• Employ 2D blue native-polyacrylamide gel electrophoresis (BN-PAGE) for complex separation

• Perform co-immunoprecipitation studies with both GLIPR1L1 and IZUMO1 antibodies

• Validate using both reducing and native conditions in Western blotting

Research has demonstrated that GLIPR1L1 forms at least six predominant complexes of ~200-1000 kDa, with specific interactions in oolemmal protein-binding complexes . Different GLIPR1L1 isoforms (37, 47, and 32 kDa) associate with distinct complexes - the 47-kDa isoform predominantly associates with complex I, while the 37- and 32-kDa isoforms predominantly associate with complex IV .

For reciprocal validation, co-immunoprecipitation using IZUMO1 antibody from acrosome-reacted spermatozoa should pull down GLIPR1L1, and vice versa .

What methods are recommended for investigating GLIPR1L1 function in fertilization using knockout models?

When developing GLIPR1L1 knockout models for fertility research, consider these methodological approaches:

• CRISPR-Cas9 gene editing targeting exon 1, which can result in a frameshift mutation and premature stop codon

• Validate knockout through both mRNA expression (quantitative PCR showing >90% reduction) and protein analysis (immunofluorescent labeling)

• Assess male fertility parameters including:

  • Sperm count and morphology

  • Sperm-zona pellucida binding assays

  • In vitro fertilization rates

  • Acrosome reaction competence

Previous research created a Glipr1l1 null mouse line with a 7-bp deletion in exon 1, resulting in a premature stop codon. This mutation caused a 92% reduction in testis mRNA expression and absence of GLIPR1L1 protein as determined by immunofluorescent labeling . This model allowed investigation of GLIPR1L1's role in optimal fertilization.

How can computational modeling approaches improve GLIPR1L1 antibody design and affinity?

Advanced computational approaches can significantly enhance GLIPR1L1 antibody design through:

• Rosetta-based computational prediction of binding interface mutations to improve affinity

• dTERMen, an informatics approach for optimization

• Combined phage display library screening with in silico predictions

• Log-likelihood scoring to rank antibody sequence designs based on binding potential

This approach yielded significant improvements in antibody affinity, with one study demonstrating improvement in KD from 0.63 nM (parental) to 0.01 nM for an optimized variant . Beyond improved affinity, this approach can also enhance cross-reactivity to related protein variants .

Methodological workflow:

• Generate a structural model of the antibody-antigen complex

• Predict mutations that could improve binding using computational algorithms

• Incorporate predictions into a phage display library

• Screen library for binding affinity

• Validate top candidates through SPR binding assays

What is the relationship between GLIPR1L1 and GLIPR1 in research applications, and how can antibody cross-reactivity be assessed?

GLIPR1L1 is part of a gene family that includes GLIPR1, a tumor suppressor protein. While they share structural similarities, their functions differ significantly:

• GLIPR1L1: Primarily involved in sperm-egg binding and fertilization

• GLIPR1: Functions as a tumor suppressor, particularly in prostate cancer

When using antibodies for either protein, cross-reactivity assessment is critical:

• Test antibodies against recombinant proteins of both GLIPR1L1 and GLIPR1

• Validate specificity using tissues with differential expression (GLIPR1L1 is highly expressed in testis, while GLIPR1 expression patterns differ)

• Include knockout controls where available

• Verify epitope specificity - most commercial GLIPR1L1 antibodies are raised against amino acid regions 20-70

What methodologies are optimal for studying post-translational modifications of GLIPR1L1 in relation to antibody recognition?

Post-translational modifications (PTMs) of GLIPR1L1 can significantly affect antibody recognition and protein function. To investigate these relationships:

• Use glycosylation-specific detection methods (lectins, glycosidase treatments) before antibody application

• Employ mass spectrometry to characterize PTMs on immunoprecipitated GLIPR1L1

• Apply multivariate quantitative analysis techniques similar to those used for IgG1 effector function studies

• Compare antibody recognition patterns in tissues with differential PTM machinery

Research has demonstrated that even small changes in glycosylation can dramatically affect protein-protein interactions, as observed with IgG1 where a 1% decrease in Fc fucosylation led to a >25% increase in antibody-dependent cell-mediated cytotoxicity . Similar sensitivity may exist for GLIPR1L1 modifications.

Experimental approach: Compare GLIPR1L1 antibody recognition patterns in samples before and after treatment with PTM-modifying enzymes (phosphatases, glycosidases, etc.) to determine how modifications affect epitope accessibility and complex formation.

How should researchers optimize Western blot protocols specifically for GLIPR1L1 detection?

Optimizing Western blot protocols for GLIPR1L1 requires specific considerations:

For difficult-to-detect isoforms or complexes, consider:

• Extended transfer times for high molecular weight complexes

• Use of specialized membrane types (PVDF for high sensitivity)

• Signal enhancement systems for low abundance detection

What are the critical parameters for reproducible immunohistochemistry with GLIPR1L1 antibodies?

Reproducible immunohistochemistry for GLIPR1L1 requires attention to several critical parameters:

• Antigen retrieval method: Evidence suggests TE buffer pH 9.0 provides optimal epitope accessibility; citrate buffer pH 6.0 may be used as an alternative

• Antibody dilution ranges:

  • For research-grade IHC: 1:20-1:200

  • For high-sensitivity applications: 1:1000-1:2500

• Positive control selection: Human testis tissue provides the most reliable positive control for GLIPR1L1 expression

• Detection system: HRP-based systems with DAB substrate provide good signal-to-noise ratio for GLIPR1L1 detection

• Counterstaining: Light hematoxylin counterstain to avoid obscuring specific signal

For multiplex applications combining GLIPR1L1 with other markers, sequential immunostaining with appropriate blocking steps between antibodies is recommended to prevent cross-reactivity.

How can researchers resolve contradictory results when using different GLIPR1L1 antibodies?

When faced with contradictory results using different GLIPR1L1 antibodies, implement this systematic troubleshooting approach:

• Compare epitope regions: Different antibodies target different regions of GLIPR1L1

  • N-terminal antibodies (amino acids 20-70)

  • Mid-region antibodies

  • C-terminal antibodies

• Evaluate antibody formats and classes:

  • Polyclonal vs. monoclonal (recombinant monoclonals typically show higher consistency)

  • Host species (rabbit antibodies predominate for GLIPR1L1)

• Validation with orthogonal methods:

  • Confirm with RNA expression data

  • Use knockout/knockdown controls

  • Verify with mass spectrometry

• Isoform specificity assessment:

  • Some antibodies detect both known isoforms while others may be isoform-specific

  • Different isoforms associate with distinct protein complexes

• Technical validation:

  • Use multiple antibodies targeting different epitopes

  • Perform blocking peptide experiments

  • Compare recombinant vs. native protein detection