lrrc57 Antibody

What validation methods should researchers employ to confirm LRRC57 antibody specificity?

Proper validation of LRRC57 antibodies requires a multi-faceted approach:

• Knockout/knockdown validation: Use of LRRC57 knockout cell lines represents the gold standard for antibody validation. The YCharOS study demonstrated that knockout cell lines serve as superior controls for antibody validation, particularly for Western blots and immunofluorescence imaging .

• Multi-application testing: Verify antibody performance across different applications:

  • Western blotting with predicted band size of 27 kDa

  • Immunohistochemistry with appropriate tissue controls

  • Immunofluorescence with subcellular localization assessment

  • Flow cytometry with proper gating strategies

• Cross-reactivity assessment: Test for potential cross-reactivity with other LRRC family members (LRRC56, LRRC55, LRRC53, etc.) which share structural similarity with LRRC57 .

• Multiple antibody concordance: Compare results from antibodies targeting different epitopes of LRRC57 to ensure consistent detection patterns.

• Peptide competition assays: Perform competition assays with specific peptides to confirm binding specificity.

According to the YCharOS study, approximately 12 publications per protein target included data from antibodies that failed to recognize the relevant target protein, highlighting the critical importance of rigorous validation .

What are the optimal conditions for using LRRC57 antibodies in Western blot applications?

Western blot optimization for LRRC57 detection requires careful consideration of several parameters:

For optimal results:

• Include both positive controls (recombinant LRRC57) and negative controls (LRRC57 knockout/knockdown samples)

• Perform titration experiments to determine optimal antibody concentration

• Use freshly prepared samples with protease inhibitors to prevent degradation

• Consider gradient gels for better resolution around the 27 kDa range

What are the advantages and limitations of different antibody types for LRRC57 research?

Based on current research in antibody technology, including information from the YCharOS study , researchers should consider the following characteristics when selecting an LRRC57 antibody type:

The YCharOS study demonstrated that recombinant antibodies outperformed both monoclonal and polyclonal antibodies across multiple assays , suggesting they may be the optimal choice for LRRC57 research when available and budget allows.

How can researchers address false negative results when using LRRC57 antibodies?

False negative results with LRRC57 antibodies may stem from multiple sources. The following systematic approach can help identify and resolve issues:

• Sample preparation issues:

  • Ensure complete protein extraction with appropriate lysis buffers

  • Add protease inhibitors to prevent LRRC57 degradation

  • Avoid excessive sample heating which may denature epitopes

  • Consider non-denaturing conditions if targeting conformational epitopes

• Technical parameters:

  • Optimize antibody concentration through titration (1:200-1:4000 for WB )

  • Extend primary antibody incubation time (overnight at 4°C)

  • Adjust detection method sensitivity (chemiluminescence vs. fluorescence)

  • For IHC, optimize antigen retrieval methods (observed at 1:40 dilution in thyroid cancer tissue )

• Epitope accessibility:

  • Consider the LRR structure of LRRC57 and potential epitope masking

  • Try alternative antibodies targeting different regions of LRRC57

  • For fixed samples, test different fixation methods that may better preserve epitopes

  • Consider membrane permeabilization optimization for intracellular detection

• Expression level considerations:

  • Verify LRRC57 expression in your biological system using RT-qPCR

  • Use positive control lysates (HT-29, HepG2, or Jurkat cells )

  • Consider enrichment by immunoprecipitation before detection

  • Evaluate potential post-translational modifications affecting antibody binding

• Verification approaches:

  • Test multiple antibodies against different LRRC57 epitopes

  • Use recombinant LRRC57 protein as positive control

  • Consider tagged LRRC57 expression systems for verification

What are the optimal methods for co-immunoprecipitation studies involving LRRC57?

Co-immunoprecipitation (Co-IP) of LRRC57 requires special considerations due to its leucine-rich repeat structure, which mediates protein-protein interactions:

• Antibody selection for Co-IP:

  • Choose antibodies validated specifically for immunoprecipitation

  • Select antibodies recognizing native (non-denatured) LRRC57

  • Consider using antibodies that target regions away from potential interaction domains

  • Alternative approach: Use tagged LRRC57 constructs and anti-tag antibodies

• Lysis conditions optimization:

  • Use mild lysis buffers to preserve protein-protein interactions

  • Test different detergents: NP-40 (0.5-1%), Triton X-100 (0.5-1%), or CHAPS (0.3-1%)

  • Include protease and phosphatase inhibitor cocktails

  • Optimize salt concentration (typically 100-150 mM NaCl)

  • Maintain pH between 7.2-7.5 to preserve interactions

• Protocol considerations:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Pre-form antibody-bead complexes before adding lysate

  • Include gentle agitation (rotation rather than shaking)

  • Optimize incubation time and temperature (4°C overnight typically works well)

  • Use stringent but non-denaturing wash conditions

• Controls to include:

  • Input lysate (5-10% of IP sample)

  • IgG control from same species as LRRC57 antibody

  • LRRC57 knockout/knockdown negative control

  • Reciprocal IP when possible

  • Isotype controls to assess non-specific binding

• Results validation:

  • Confirm LRRC57 in immunoprecipitates by Western blot

  • Consider mass spectrometry to identify novel interacting partners

  • Validate key interactions with orthogonal methods (proximity ligation, FRET)

  • Assess interaction under different cellular conditions

Since LRRC57 contains multiple leucine-rich repeats, it likely participates in various protein-protein interactions , making Co-IP a valuable approach for elucidating its functional roles.

How does LRRC57 conservation across species impact antibody selection?

LRRC57 is described as "exceedingly well conserved" across species , which has significant implications for antibody selection and experimental design:

• Cross-species reactivity profile:

  • Many LRRC57 antibodies demonstrate cross-reactivity with human, mouse, and rat LRRC57

  • Antibody ABIN7237338 shows reactivity with human, mouse, and rat samples

  • ABIN1630981 is specifically designed for rat LRRC57 detection

• Epitope conservation analysis:

  • High sequence conservation suggests epitopes may be preserved across species

  • Allows for translational research across model organisms

  • May complicate generation of species-specific antibodies

  • Enables validation across multiple species as a specificity check

• Strategic considerations:

  • When species-specificity is required, target less conserved regions

  • Validate cross-reactivity experimentally rather than relying solely on sequence homology

  • Consider using cross-reactive antibodies to compare LRRC57 function across species

  • Leverage conservation for validation (consistent molecular weight, localization)

• Experimental validation approaches:

  • Test antibodies on samples from multiple species under identical conditions

  • Confirm similar band patterns in Western blot applications

  • Verify consistent subcellular localization in imaging studies

  • Use species-specific knockout/knockdown controls

This high conservation suggests LRRC57 may perform important biological functions preserved throughout evolution, making comparative studies across species particularly valuable.

How might post-translational modifications of LRRC57 affect antibody binding?

While specific post-translational modifications (PTMs) of LRRC57 are not extensively documented in the provided search results, their potential impact on antibody binding must be considered:

• Potential PTMs affecting LRRC57 detection:

  • Phosphorylation of serine/threonine residues in signaling contexts

  • Ubiquitination affecting protein stability and turnover

  • Glycosylation potentially affecting protein folding or interaction

  • Acetylation or methylation of lysine residues

  • SUMOylation impacting protein localization

• Impact on antibody binding:

  • Epitope masking: PTMs directly within antibody binding sites

  • Conformational changes: PTMs altering protein folding and tertiary structure

  • Creation of neo-epitopes: Modified residues becoming part of new antigenic determinants

  • Altered protein interactions: PTMs affecting complex formation and accessibility

• Experimental strategies:

  • Use multiple antibodies targeting different epitopes

  • Compare results from different cellular conditions that may alter PTM status

  • Consider phospho-specific antibodies if phosphorylation sites are characterized

  • Treat samples with enzymes that remove specific PTMs (phosphatases, glycosidases)

  • Use mass spectrometry to identify and map PTMs on LRRC57

• Documentation and controls:

  • Record cell treatment conditions that might alter PTM status

  • Include appropriate controls for PTM-inducing conditions

  • Consider how sample preparation methods affect PTM preservation

  • Compare results across different detection methods

Given LRRC57's potential roles in signaling and its leucine-rich repeat structure, it's reasonable to hypothesize that PTMs might regulate its function and interactions, potentially affecting antibody recognition in experimental contexts.

How are new antibody generation technologies improving LRRC57 research?

Recent advances in antibody technology have significant implications for LRRC57 research, as demonstrated by several sources in the search results:

• Phage display and recombinant antibody technologies:

  • Search result describes "inference and design of antibody specificity" using phage display experiments

  • This approach allows identification of distinct binding modes for specific ligands

  • The technology enables prediction and generation of specific variants beyond those observed experimentally

  • Particularly valuable for designing antibodies with both specific and cross-specific binding properties

• Golden Gate-based dual-expression systems:

  • Search result details a new genotype-phenotype linked antibody screening system

  • This method enables rapid isolation of antibodies within 7 days

  • The system links heavy-chain and light-chain variable DNA fragments from single-sorted B cells

  • Membrane-bound Ig expression allows flow cytometry-based selection of high-affinity antibodies

• Biophysics-informed computational modeling:

  • Integration of experimental data with computational models to predict antibody specificity

  • Enables disentangling of multiple binding modes associated with specific targets

  • Allows generation of antibodies with customized specificity profiles

  • Can mitigate experimental artifacts and biases in selection experiments

• High-throughput antibody characterization initiatives:

  • The YCharOS initiative systematically analyzes antibody performance

  • Their study of 614 antibodies targeting 65 proteins revealed that 50-75% of proteins had at least one high-performing commercial antibody

  • Industry-researcher partnerships improved antibody quality and application recommendations

  • Demonstrated superior performance of recombinant antibodies compared to traditional monoclonal and polyclonal antibodies

These technological advances offer promising avenues for developing higher quality, more specific LRRC57 antibodies for various research applications.

How should researchers systematically evaluate and compare commercial LRRC57 antibodies?

Based on principles outlined in the antibody characterization literature , researchers should implement a structured approach to evaluate commercial LRRC57 antibodies:

• Standardized multi-application assessment:

  • Test antibodies in multiple applications (WB, IHC, IF, ELISA) under identical conditions

  • Document performance metrics for each application

  • Compare results across applications to assess versatility

• Validation with appropriate controls:

  • LRRC57 knockout cell lines provide the strongest negative controls

  • Recombinant LRRC57 protein serves as positive control

  • Cell lines with verified LRRC57 expression (HT-29, HepG2, Jurkat)

  • Isotype controls to assess non-specific binding

• Quantitative performance metrics:

  • Signal-to-noise ratio across applications

  • Limit of detection with dilution series

  • Dynamic range of detection

  • Reproducibility across replicates and lots

  • Correlation with orthogonal measures of LRRC57 expression

• Cross-reactivity assessment:

  • Test against other LRRC family members (LRRC56, LRRC55, etc.)

  • Evaluate species cross-reactivity claims experimentally

  • Assess potential cross-reactivity with proteins of similar size

• Documentation and reporting standards:

  • Maintain detailed records of optimization conditions

  • Document lot numbers and batch information

  • Report dilution factors yielding equivalent signal intensity

  • Compare exposure times needed for comparable results

The YCharOS study demonstrated that commercial catalogs contain specific and renewable antibodies for more than half of the human proteome , but systematic evaluation remains essential to identify the most suitable LRRC57 antibodies for specific research applications.