LCP1 Recombinant Monoclonal Antibody

What is LCP1 and why is it important in research?

LCP1 (also known as Plastin L) is an actin-binding protein that plays a crucial role in the organization and regulation of the actin cytoskeleton. Its functions extend to influencing cell migration, immune cell activation, microvilli formation, cancer metastasis, neuronal processes, and regulation of immune responses. The protein's ability to crosslink actin filaments contributes to cellular structure and function in various cell types . Due to its involvement in multiple cellular processes, LCP1 is an important research target for understanding cytoskeletal dynamics, immune cell function, and cancer progression.

How are LCP1 recombinant monoclonal antibodies generated?

LCP1 recombinant monoclonal antibodies are generated through in vitro processes using synthetic genes. The production follows these steps: First, LCP1 antibody genes are isolated from B cells sourced from immunoreactive rabbits. These genes are then amplified and cloned into appropriate phage vectors. The vectors are subsequently transfected into mammalian cell lines to enable the production of functional antibodies. Finally, the LCP1 recombinant monoclonal antibody is purified from the culture supernatant of the transfected cell lines through affinity chromatography . This process ensures consistent antibody production with high specificity and reproducibility compared to traditional hybridoma methods.

What are the typical applications for LCP1 recombinant monoclonal antibodies?

LCP1 recombinant monoclonal antibodies demonstrate good results in multiple applications:

These applications allow researchers to study LCP1 expression, localization, and function in various experimental systems . Flow cytometry is particularly useful for analyzing LCP1 expression in immune cells, while IHC provides insights into its distribution in tissues such as lymphoid organs.

How does LCP1 recombinant monoclonal antibody performance compare to traditional antibodies in complex experimental systems?

Recombinant monoclonal antibodies offer several advantages over traditional hybridoma-derived antibodies in complex experimental systems. Unlike hybridoma-derived antibodies, recombinant antibodies show consistent lot-to-lot reproducibility since they are produced from known sequences in controlled expression systems . This results in more reliable experimental outcomes, especially in longitudinal studies where antibody consistency is critical.

When specifically examining LCP1 detection, recombinant antibodies demonstrate high specificity across multiple applications. For example, in flow cytometry analyses of complex cell populations, the LCP1 recombinant monoclonal antibody effectively distinguishes target cells with minimal background, as evidenced by the clear separation between control and stained populations in U20S cells . Similarly, in immunohistochemistry studies of human tonsil and B lymphocytic tumor tissues, the antibody produces precise localization with minimal non-specific binding . These characteristics make recombinant antibodies particularly valuable for multiparameter experiments where signal specificity is essential.

What are the critical validation steps needed when using LCP1 recombinant monoclonal antibodies in new experimental contexts?

When employing LCP1 recombinant monoclonal antibodies in new experimental contexts, several validation steps are critical:

• Positive and Negative Controls: Include known LCP1-expressing cells/tissues (e.g., HL-60, THP-1, spleen tissues) as positive controls and known LCP1-negative samples as negative controls .

• Antibody Titration: Perform dilution series experiments (e.g., 1:50, 1:100, 1:200 for flow cytometry) to determine optimal antibody concentration for your specific sample type .

• Cross-Reactivity Testing: Verify specificity by testing the antibody against related proteins or in samples from different species if cross-reactivity is claimed.

• Blocking Experiments: Conduct peptide blocking experiments using the immunogen peptide to confirm binding specificity.

• Orthogonal Method Validation: Confirm results using alternative detection methods (e.g., if using FC, validate with WB or IHC).

• Knockout/Knockdown Validation: When possible, test the antibody in LCP1 knockout or knockdown systems to confirm specificity.

These validation steps ensure that experimental findings reflect true LCP1 biology rather than artifacts or non-specific interactions, particularly important when studying LCP1 in novel cell types or experimental conditions.

How can researchers optimize fixation and permeabilization protocols for LCP1 detection in different cell types?

Optimization of fixation and permeabilization protocols is critical for accurate LCP1 detection, as this cytoskeletal protein requires careful sample preparation to maintain epitope accessibility while preserving cellular architecture. Based on published protocols, the following optimization strategies are recommended:

For flow cytometry applications:

• Fixation: 4% paraformaldehyde provides good results while maintaining cellular integrity. Fix cells for 10-15 minutes at room temperature .

• Permeabilization: 0.2% TritonX-100 is effective for accessing intracellular LCP1. For more sensitive cell types, consider milder alternatives like 0.1% saponin .

• Blocking: Use 10% normal goat serum to reduce non-specific binding before antibody incubation .

• Incubation Conditions: Optimal results are achieved with primary antibody incubation at 4°C for 30-45 minutes at concentrations of 1μg per 10^6 cells .

For immunohistochemistry of tissues:

• Antigen Retrieval: Heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes significantly improves LCP1 detection in paraffin-embedded sections .

• Section Thickness: 5-7μm sections provide optimal balance between structural integrity and antibody penetration.

• Protocol Modification: For tissues with high endogenous peroxidase activity, include an additional peroxidase quenching step.

These parameters should be systematically tested and adjusted based on the specific cell type or tissue being examined, as LCP1 accessibility can vary significantly between different sample types.

What are the optimal storage and handling conditions for LCP1 recombinant monoclonal antibodies to maintain functionality?

To maintain optimal functionality of LCP1 recombinant monoclonal antibodies, proper storage and handling are essential:

Storage Conditions:

• Store antibodies at -20°C or -80°C for long-term preservation .

• Avoid repeated freeze-thaw cycles, which can denature antibodies and reduce activity .

• For working solutions, store at 4°C for up to two weeks.

• The antibody is typically provided in a stabilizing buffer containing phosphate buffered saline (pH 7.4), 150mM NaCl, 0.02% sodium azide, and 50% glycerol .

Handling Recommendations:

• Thaw antibodies completely but gently at 4°C before use.

• Centrifuge briefly before opening vials to collect all liquid at the bottom.

• Use sterile technique when handling to prevent contamination.

• Prepare working dilutions shortly before use and discard unused diluted antibody.

• Transport on ice when moving between laboratory locations.

Aliquoting Strategy:

• Upon first receipt, consider dividing the antibody into small single-use aliquots to minimize freeze-thaw cycles.

• Document lot numbers and preparation dates for each aliquot.

Following these guidelines ensures that the LCP1 recombinant monoclonal antibody maintains its specificity and sensitivity throughout the experimental timeline, leading to more consistent and reliable results.

How can researchers troubleshoot weak or non-specific signals when using LCP1 antibodies in flow cytometry?

When encountering weak or non-specific signals in flow cytometry experiments with LCP1 antibodies, systematic troubleshooting is essential:

For Weak Signals:

• Antibody Concentration: Increase antibody concentration within recommended range (1:50-1:200). Consider testing a titration series to determine optimal concentration .

• Incubation Conditions: Extend primary antibody incubation time from 30-45 minutes to overnight at 4°C. Ensure consistent temperature throughout incubation .

• Fixation/Permeabilization Optimization: Modify permeabilization protocol to improve antibody access to intracellular epitopes. Try increasing Triton X-100 concentration from 0.2% to 0.3%, or test alternative permeabilization agents .

• Secondary Antibody Enhancement: For indirect detection methods, use a more sensitive secondary antibody or increase its concentration. For example, adjust FITC-conjugated Goat Anti-rabbit IgG(H+L) from 1:200 to 1:100 .

• Sample Preparation: Ensure cells are properly dissociated and filtered to prevent clumping which can reduce signal detection.

For Non-Specific Signals:

• Blocking Optimization: Increase normal serum blocking from 10% to 15-20% to reduce background. Consider adding 0.1% BSA to blocking and washing buffers .

• Washing Protocol: Implement more stringent washing steps between antibody incubations. Increase wash volume and number of washes (3-5 times with larger volumes).

• Control Antibody Comparison: Compare staining pattern with isotype control (rabbit IgG at equivalent concentration) to identify non-specific binding .

• Dead Cell Exclusion: Include viability dye to exclude dead cells which often produce autofluorescence and non-specific binding.

• FcR Blocking: Add Fc receptor blocking reagent if using immune cells known to express high levels of Fc receptors.

By systematically addressing these parameters, researchers can optimize LCP1 detection in flow cytometry experiments, ensuring specific and robust signal detection.

What are the recommended sample preparation protocols for detecting LCP1 in different tissue types?

Sample preparation protocols must be tailored to specific tissue types for optimal LCP1 detection. Based on validated methods, the following tissue-specific recommendations apply:

For Lymphoid Tissues (Spleen, Tonsil, Lymph Nodes):

• Fixation: 10% neutral buffered formalin for 24-48 hours, followed by paraffin embedding .

• Sectioning: 5μm sections on positively charged slides.

• Antigen Retrieval: Heat-mediated retrieval in citrate buffer (pH 6.0) for 20 minutes at 95-98°C .

• Blocking: 10% goat serum for 1 hour at room temperature to block non-specific binding .

• Antibody Incubation: 1μg/ml anti-LCP1 antibody overnight at 4°C .

• Detection System: Streptavidin-biotin complex with DAB chromogen provides excellent signal-to-noise ratio in lymphoid tissues .

For Tumor Tissues:

• Fixation: Shorter fixation times (12-24 hours) to preserve epitope accessibility.

• Additional Blocking: Include 0.3% hydrogen peroxide treatment to quench endogenous peroxidase activity before antibody incubation.

• Background Reduction: Add 0.1% Triton X-100 to washing buffers to reduce non-specific binding common in tumor tissues .

• Control Sections: Adjacent sections stained with isotype control are particularly important for tumors to distinguish specific staining from tumor-associated non-specific binding.

For Cell Line Samples:

• Cell Preparation: For Western blot analysis, 30μg of protein lysate per lane provides optimal detection .

• Lysis Buffer: RIPA buffer supplemented with protease inhibitors effectively extracts LCP1 while preserving protein integrity.

• Flow Cytometry Preparation: Fix cells with 4% paraformaldehyde and permeabilize with permeabilization buffer before blocking with 10% normal goat serum .

These tissue-specific protocols have been validated for LCP1 detection and should be further optimized based on the specific research question and experimental system.

How can researchers differentiate between LCP1 isoforms using recombinant monoclonal antibodies?

Differentiating between LCP1 isoforms requires careful selection and application of recombinant monoclonal antibodies:

Epitope Selection Strategy:
The LCP1 recombinant monoclonal antibody (CSB-RA188599A0HU) is generated using a synthesized peptide derived from human LCP1 as the immunogen . To differentiate between isoforms, researchers should determine which epitope region the antibody recognizes by consulting the manufacturer's epitope mapping data. If the epitope lies within a region that differs between isoforms, the antibody may inherently distinguish them.

Recommended Experimental Approach:

• High-Resolution Western Blotting: Use 5-20% gradient SDS-PAGE gels run at lower voltage (70-90V) for extended periods (2-3 hours) to achieve maximum separation of closely related isoforms . This approach has successfully distinguished LCP1 at approximately 70 kDa in various cell lysates.

• Two-Dimensional Electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate isoforms based on both charge and molecular weight differences.

• Immunoprecipitation Followed by Mass Spectrometry: Use the LCP1 antibody to immunoprecipitate the protein, then identify specific isoforms through mass spectrometry analysis.

• Comparative Analysis: When studying multiple samples, comparative analysis across different cell types with known isoform expression patterns (e.g., HL-60, THP-1, and HeLa cells) can provide reference points for isoform identification .

• Knockout/Knockdown Validation: Generate or obtain cells with specific LCP1 isoforms knocked out or down to serve as negative controls for particular isoforms.

These approaches, particularly when used in combination, provide researchers with robust methods for distinguishing between LCP1 isoforms, enabling more precise analysis of isoform-specific functions in different biological contexts.

What are the considerations for using LCP1 recombinant monoclonal antibodies in multiplexed imaging or flow cytometry experiments?

When incorporating LCP1 recombinant monoclonal antibodies into multiplexed imaging or flow cytometry experiments, several critical considerations ensure optimal results:

Spectral Compatibility and Panel Design:

• Fluorophore Selection: Since the LCP1 antibody (CSB-RA188599A0HU) is non-conjugated , researchers must select secondary antibodies with fluorophores that minimize spectral overlap with other channels in the panel.

• Secondary Antibody Species: Using rabbit-derived LCP1 antibodies requires anti-rabbit secondary antibodies, necessitating careful planning to avoid cross-reactivity with other primary antibodies in the panel.

• Signal Intensity Balancing: Adjust antibody concentrations to balance signal intensities across different markers, particularly important when LCP1 shows strong expression (as seen in RAJI cells) .

Protocol Optimization for Multiplexing:

• Sequential Staining: For challenging multiplex panels, consider sequential staining approaches where LCP1 detection is performed separately from potentially cross-reactive antibodies.

• Universal Fixation/Permeabilization: Identify fixation and permeabilization conditions compatible with all antibodies in the panel. The 4% formaldehyde fixation with 0.2% TritonX-100 permeabilization used for LCP1 may need adjustment to accommodate other targets.

• Blocking Strategy: Use a comprehensive blocking approach with 10% normal goat serum supplemented with Fc receptor blockers when working with immune cells.

Validation for Multiplexed Applications:

• Single-Stain Controls: Mandatory for each antibody in the panel for compensation calculation.

• Fluorescence Minus One (FMO) Controls: Essential for establishing gating boundaries, particularly for dim LCP1 expression.

• Cross-Reactivity Testing: Verify that secondary antibodies do not cross-react with other primary antibodies in the panel.

Data Analysis Considerations:

• Compensation Matrices: Careful compensation is critical when LCP1 signal is strong and may spill into adjacent channels.

• Co-expression Analysis: When analyzing LCP1 co-expression with other markers, use bivariate plots with proper compensation to accurately assess co-expression patterns.

These considerations ensure that LCP1 recombinant monoclonal antibodies can be effectively integrated into complex multiplexed experimental designs while maintaining specificity and sensitivity.

How can researchers effectively use LCP1 recombinant monoclonal antibodies to study LCP1's role in cancer and immune regulation?

LCP1's involvement in both cancer progression and immune system regulation makes it a valuable target for translational research. Researchers can effectively leverage LCP1 recombinant monoclonal antibodies through the following methodological approaches:

For Cancer Research Applications:

• Expression Profiling: Using IHC with anti-LCP1 antibodies on tissue microarrays to evaluate LCP1 expression across different cancer types and stages. The validated protocol for B lymphocytic tumor tissue can be adapted for various cancer tissues.

• Metastasis Studies: Employing flow cytometry to quantify LCP1 expression in circulating tumor cells, using the validated FC protocol (1:50-1:200 dilution) combined with other cancer stem cell markers.

• Mechanistic Investigations: Performing co-immunoprecipitation with LCP1 antibodies to identify cancer-specific interaction partners that may reveal mechanistic insights into LCP1's role in cancer cell migration and invasion.

• Therapy Response Monitoring: Using LCP1 antibodies to track changes in expression and localization following treatment, potentially identifying it as a biomarker for therapy response.

For Immune Regulation Studies:

• Immune Cell Phenotyping: Implementing multiparameter flow cytometry panels including LCP1 to characterize its expression across immune cell subsets. The validated staining of RAJI cells provides a foundation for this approach.

• Functional Correlation Studies: Correlating LCP1 expression levels (quantified by flow cytometry) with functional assays such as migration, cytokine production, or cytotoxicity to establish functional significance.

• Spatial Distribution Analysis: Using immunofluorescence with LCP1 antibodies to analyze its distribution within immune tissues and at immune synapses, building on validated IHC protocols for tonsil tissues .

• Dynamic Regulation Studies: Employing time-course experiments with LCP1 antibodies to track changes in expression and localization following immune stimulation or suppression.

Integrated Research Approaches:

• Tumor Microenvironment Analysis: Combining LCP1 staining with immune cell markers in multiplex IHC to examine interactions between cancer cells and immune cells within the tumor microenvironment.

• Patient-Derived Models: Applying validated LCP1 antibody protocols to patient-derived xenografts or organoids to study LCP1's role in a clinically relevant context.

These methodological approaches enable researchers to effectively utilize LCP1 recombinant monoclonal antibodies to unravel LCP1's multifaceted roles in cancer progression and immune regulation, potentially identifying new therapeutic targets or biomarkers.