LCR55 Antibody

What is LRRC55 and what is the function of LRRC55 antibodies in research?

LRRC55 (Leucine-rich repeat-containing protein 55) functions as an auxiliary protein of the large-conductance, voltage and calcium-activated potassium channel (BK alpha). It modulates gating properties by producing a marked shift in the BK channel's voltage dependence of activation in the hyperpolarizing direction, and functions in the absence of calcium . LRRC55 antibodies are critical research tools used to:

• Detect and quantify LRRC55 protein expression in various tissues and cells

• Characterize the subcellular localization of LRRC55 in relation to ion channels

• Study protein-protein interactions involving LRRC55

• Investigate the role of LRRC55 in neurological and physiological processes

For neuroscience applications, these antibodies are particularly valuable for studying potassium channel regulation in neuronal excitability and function.

What methods are recommended for validating the specificity of LRRC55 antibodies?

Antibody validation using knockout (KO) cell lines is now considered the gold standard approach for ensuring specificity. A comprehensive validation should include:

The YCharOS initiative has developed standardized antibody validation procedures showing that "using a parental cell line that expresses the target protein, alongside an isogenic CRISPR knockout version of the same cell, provides rigorous and broadly applicable results" . When selecting a cell line for LRRC55 validation, researchers should prioritize lines with RNA expression levels above 2.5 log2(TPM+1) to ensure sufficient protein expression .

How should I select the right LRRC55 antibody for my specific application?

When selecting an LRRC55 antibody, consider these critical factors:

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

• Epitope location: Consider which region of LRRC55 the antibody targets (e.g., ab121412 targets aa 100-250)

• Validation data quality: Look for antibodies validated using knockout controls rather than just overexpression systems

• Clonality: Determine whether a monoclonal (consistent, specific) or polyclonal (broader epitope recognition) is more appropriate

• Species reactivity: Ensure cross-reactivity with your species of interest

• Citation record: Check if the antibody has been successfully used in published research

Recent studies emphasize that "a combination of a biochemically purified F1 protein and recombinant V-antigen provided more protection than the individual proteins" when studying antibody interactions , suggesting that carefully considering epitope regions is critical for antibody selection.

What are the optimal protocols for using LRRC55 antibodies in immunohistochemistry?

Based on available data for LRRC55 antibodies in immunohistochemistry:

Recommended Protocol for Paraffin-Embedded Sections:

• Deparaffinization and rehydration: Standard xylene and ethanol gradient

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

• Blocking: 5% normal serum in PBS with 0.1% Triton X-100 for 1 hour at room temperature

• Primary antibody: Dilute LRRC55 antibody 1:35 (as reported for ab121412) in blocking buffer

• Incubation: Overnight at 4°C in a humidified chamber

• Detection: HRP-conjugated secondary antibody followed by DAB substrate

• Counterstaining: Hematoxylin for nuclear visualization

For human stomach tissue, this protocol has shown strong cytoplasmic positivity in parietal cells using LRRC55 antibody . Different tissue types may require optimization of antibody concentration and antigen retrieval methods.

What considerations are important when using LRRC55 antibodies for Western blotting?

For optimal Western blot results with LRRC55 antibodies:

• Sample preparation: Use non-reducing conditions as some LRRC55 epitopes may be sensitive to reducing agents

• Expected molecular weight: Look for bands at approximately 60-75 kDa (variable due to potential post-translational modifications)

• Blocking optimization: Test both BSA and milk-based blockers as some antibodies perform better with specific blocking agents

• Membrane selection: PVDF membranes are recommended for optimal protein binding and signal detection

• Transfer conditions: Use wet transfer at 30V overnight at 4°C for large proteins

• Antibody dilution: Start with manufacturer's recommended dilution (e.g., 2 μg/mL) and optimize as needed

• Visualization: HRP-conjugated secondary antibodies with enhanced chemiluminescence detection systems

As demonstrated with other antibodies: "Western blot shows lysates of mouse spleen tissue and mouse splenocytes. PVDF membrane was probed with 2 μg/mL of antibody followed by HRP-conjugated Anti-Rat IgG Secondary Antibody. A specific band was detected at approximately 75 kDa. This experiment was conducted under non-reducing conditions and using Immunoblot Buffer Group 1. No bands were observed when using reducing conditions" .

How can I troubleshoot inconsistent results when using LRRC55 antibodies?

When facing inconsistent results with LRRC55 antibodies, implement this systematic troubleshooting approach:

Research has shown that "it is commonly understood that many commercial antibodies do not recognize their intended targets" , making systematic validation and troubleshooting essential.

How can I assess cross-reactivity of LRRC55 antibodies with other leucine-rich repeat-containing proteins?

To comprehensively evaluate potential cross-reactivity:

• Sequence analysis: Perform bioinformatic analysis of epitope conservation across the LRR protein family

• Knockout validation: Compare staining patterns between wildtype and LRRC55 knockout samples

• Peptide competition: Pre-incubate antibody with excess synthetic peptide corresponding to the epitope region

• Overexpression systems: Test antibody reactivity against cells overexpressing related LRR proteins

• Immunoprecipitation-mass spectrometry: Identify all proteins captured by the antibody to detect off-target binding

• Protein microarrays: Screen antibody against arrays containing multiple LRR family proteins

Studies have shown that "using an appropriately selected wild type cell and an isogenic CRISPR knockout (KO) version of the same cell as the basis for testing, yields rigorous and broadly applicable results" , making this the preferred approach for cross-reactivity assessment.

What are the considerations when using LRRC55 antibodies for immunoprecipitation and protein interaction studies?

For successful immunoprecipitation with LRRC55 antibodies:

• Extraction buffer optimization:

  • Test different lysis buffers (RIPA vs. NP-40 vs. Digitonin)

  • Include appropriate protease/phosphatase inhibitors

  • Consider native vs. denaturing conditions based on interaction stability

• Antibody coupling strategy:

  • Direct coupling to beads may preserve antibody orientation

  • Pre-clearing lysates reduces non-specific binding

  • Consider cross-linking antibody to beads to prevent co-elution

• Controls for validation:

  • Include isotype control antibody IP

  • Use LRRC55 knockout cells as negative control

  • Include input, flow-through, and IP fractions for analysis

• Co-IP considerations:

  • Gentler lysis conditions preserve protein-protein interactions

  • Shorter incubation times may capture transient interactions

  • Crosslinking may stabilize weak interactions

Research has demonstrated that "an analysis of protective mAbs that bind to V-antigen was made to assess binding sites, avidities, and affinities" , highlighting the importance of characterizing antibody-antigen interactions for optimal immunoprecipitation results.

How do structural modifications of antibodies affect their binding to LRRC55 and experimental outcomes?

Structural modifications significantly impact antibody performance and should be considered when selecting or modifying LRRC55 antibodies:

Research has shown that "Variable domain glycans are postulated to convey a selective advantage through interaction with lectins and/or microbiota" and that "N-linked glycosylation sites in the VH and VL regions have been observed in 10-25% of all serum IgG, which can contribute to both antibody stability, and modulate antigen binding" .

What methodologies enable quantitative analysis of LRRC55 expression using antibody-based techniques?

For accurate quantification of LRRC55 protein levels:

• Quantitative Western blotting:

  • Use recombinant LRRC55 protein standards to create a calibration curve

  • Implement near-infrared fluorescent secondary antibodies for broader dynamic range

  • Analyze band intensity with specialized software (ImageJ, Image Studio)

  • Include appropriate housekeeping controls for normalization

• ELISA development:

  • Design sandwich ELISA using antibodies targeting different LRRC55 epitopes

  • Establish standard curves with recombinant LRRC55 protein

  • Validate assay parameters (sensitivity, linearity, reproducibility)

  • Optimize sample dilution to ensure measurements within linear range

• Flow cytometry quantification:

  • Use antibody binding capacity (ABC) beads to convert fluorescence to molecules/cell

  • Implement proper compensation for multi-parameter analysis

  • Compare to quantitative reference standards

• Surface plasmon resonance:

  • Determine binding kinetics and affinities of anti-LRRC55 antibodies

  • "The specific kinetic interactions or affinities between full-length antigen and antibodies can be accomplished using an antibody capture approach on a Biacore SPR instrument"

How are LRRC55 antibodies being used in current neuroscience research?

LRRC55 antibodies are being applied in cutting-edge neuroscience research in several key areas:

• BK channel regulation studies: Investigating how LRRC55 modulates BK channel gating properties in different neuronal populations

• Neurological disorder research: Examining potential alterations in LRRC55 expression or localization in models of epilepsy, stroke, and neurodegenerative diseases

• Circuit-specific functions: Using LRRC55 antibodies in combination with other neuronal markers to identify cell populations with specific electrophysiological properties

• Developmental neurobiology: Tracking LRRC55 expression through different developmental stages to understand its role in neuronal maturation

These research applications benefit from highly specific antibodies validated using knockout controls, as "one of the main barriers to large-scale production of high-quality antibodies is the lack of availability of KO lines derived from cells that express detectable levels of each human protein" .

What is the current landscape of antibody validation standards for LRRC55 and similar research antibodies?

The antibody validation field is evolving rapidly with several important developments:

• Consortium-based validation initiatives:

  • YCharOS (Antibody Characterization through Open Science) has developed standardized protocols for antibody validation

  • "Academic and industry scientists collaborate on a new method to characterize research antibodies"

  • The initiative has tested approximately 1,200 antibodies against 120 protein targets

• Knockout-based validation becoming standard:

  • "Using an appropriately selected wild type cell and an isogenic CRISPR knockout (KO) version of the same cell as the basis for testing, yields rigorous and broadly applicable results"

  • "Creation of a broadly accessible biobank of bespoke KO cells for each human gene should be a priority for the community"

• Multi-application testing:

  • Comprehensive validation now requires testing in multiple applications

  • "All antibodies were tested for all three applications (except that secreted proteins were not tested in IF), independent of the antibody manufacturers' recommendations"

• Data sharing platforms:

  • "Studies are rapidly shared via the open platform ZENODO, and selected studies were published on the F1000 publication platform"

  • The Antibody Registry now contains over 2.5 million commercial antibodies with assigned RRIDs (Research Resource Identifiers)

These developments are improving the reproducibility of antibody-based research and helping researchers select appropriate LRRC55 antibodies for their specific applications.

What factors should be considered when comparing different commercially available LRRC55 antibodies?

When comparing commercial LRRC55 antibodies, consider these critical factors:

• Validation methodology:

  • Prioritize antibodies validated with knockout controls

  • Compare validation data across different applications (WB, IP, IF)

  • Assess whether validation was performed in conditions relevant to your research

• Clonality and production method:

  • Recombinant antibodies offer superior batch-to-batch consistency

  • "Most antibody manufacturers prioritized renewable antibodies. The highest priority was given to recombinant antibodies as they represent the ultimate renewable reagent"

  • Monoclonal antibodies provide consistency while polyclonals may offer higher sensitivity

• Epitope considerations:

  • Compare antibodies targeting different epitopes of LRRC55

  • Consider whether the epitope is accessible in your experimental conditions

  • Evaluate conservation of the epitope across species of interest

• Published literature:

  • Assess citation history in peer-reviewed publications

  • Look for antibodies used successfully in your specific application

  • Consider reaching out to authors who have published using the antibody

• Quantitative validation metrics:

  • Signal-to-noise ratio in relevant applications

  • Binding affinity and avidity measurements if available

  • Detection limit for low-abundance samples