RFTN2 Antibody

What is RFTN2 and What Are Its Primary Functions in Cellular Processes?

RFTN2 (Raftlin Family Member 2), also known as Raft-linking protein 2 or C2orf11, is a 501 amino acid cell membrane protein essential for lipid raft assembly and maintenance . This protein plays crucial roles in immune signaling pathways through several mechanisms:

• Upon bacterial lipopolysaccharide stimulation, RFTN2 mediates clathrin-dependent internalization of TLR4 in dendritic cells, resulting in activation of TICAM1-mediated signaling and subsequent IFNB1 production

• It may regulate B-cell antigen receptor-mediated signaling

• RFTN2 closely interacts with proteins such as LAT (Linker for Activation of T cells) and SYK (Spleen tyrosine kinase), which are essential for propagating signals through immune pathways, amplifying cellular responses to external stimuli

The gene encoding RFTN2 maps to human chromosome 2q33.1 and mouse chromosome 1 C1.2 . Understanding this protein's function provides insights into immune response regulation and potential therapeutic targets for immunological disorders.

What Applications Are RFTN2 Antibodies Commonly Used For?

RFTN2 antibodies are versatile tools employed in multiple molecular and cellular biology techniques:

Most commercially available RFTN2 antibodies are rabbit polyclonal antibodies, though they target different epitopes including N-terminal regions, C-terminal regions, or specific amino acid sequences (e.g., AA 1-300, AA 100-250) . Researchers should select antibodies based on their specific experimental requirements and target species.

What Species Reactivity Should Be Considered When Selecting an RFTN2 Antibody?

Species reactivity varies significantly among different RFTN2 antibodies, which is crucial to consider when designing experiments:

When working with model organisms, researchers should verify the conservation of the targeted epitope across species. While some antibodies show broad cross-reactivity, others are specific to human RFTN2 only. For studies involving multiple species, selecting antibodies with confirmed reactivity across the relevant species is essential to ensure consistent and comparable results .

How Should RFTN2 Antibodies Be Validated Before Use in Critical Experiments?

Proper validation of RFTN2 antibodies is essential to ensure experimental reliability and reproducibility:

• Positive Control Selection: Use tissues known to express RFTN2, such as mouse lung, brain, and testis tissues, which have been confirmed to show positive signals in Western blot analyses

• Band Verification: The predicted molecular weight of RFTN2 is approximately 55-56 kDa, with observed weights ranging from 56-60 kDa in Western blots. Verification of proper band size is essential

• Specificity Testing: Consider using:

  • Comparison with RFTN2 overexpression lysates and vector-only controls

  • Knockout or knockdown systems (siRNA) to confirm antibody specificity

• Cross-Reactivity Assessment: If working with multiple species, validate the antibody in each species independently before comparative studies

• Multiple Application Testing: If planning to use the antibody for multiple techniques (e.g., WB and IHC), validate it specifically for each application, as performance can vary between techniques

Proper validation ensures experimental results are attributable to RFTN2 specifically rather than nonspecific binding or background signals.

What Are the Optimal Storage Conditions and Handling Practices for RFTN2 Antibodies?

Proper storage and handling of RFTN2 antibodies are crucial for maintaining their performance and longevity:

Some RFTN2 antibody preparations contain 0.1% BSA in smaller sizes (20μL), which helps maintain stability . Always check manufacturer-specific recommendations, as formulations vary between suppliers.

What Controls Should Be Included When Using RFTN2 Antibodies in Western Blotting?

Proper controls are essential for ensuring valid and interpretable Western blot results with RFTN2 antibodies:

• Positive Controls:

  • Use tissues known to express RFTN2 (mouse lung, brain, or testis tissue)

  • Consider RFTN2 overexpression lysates (e.g., from mammalian HEK-293T cells transfected with RFTN2)

• Negative Controls:

  • Vector-only transfected cell lysates

  • Tissues or cells known not to express RFTN2

  • Secondary antibody-only controls to assess non-specific binding

• Loading Controls:

  • Use housekeeping proteins (β-actin, GAPDH, tubulin) to normalize protein loading

  • Consider using total protein normalization methods for more accurate quantification

• Molecular Weight Markers:

  • Essential for confirming the correct band size (predicted: 55 kDa; observed: 56-60 kDa)

• Blocking Peptide Controls (for polyclonal antibodies):

  • Pre-incubation with the immunizing peptide should abolish specific staining

When interpreting Western blot results, consider that post-translational modifications may cause RFTN2 to migrate at sizes slightly different from the predicted molecular weight based on amino acid sequence alone.

What Are the Critical Parameters for Successful Immunohistochemistry with RFTN2 Antibodies?

Successful immunohistochemistry (IHC) with RFTN2 antibodies requires careful attention to several parameters:

• Tissue Preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used for RFTN2 IHC

  • Optimal fixation time affects epitope preservation

• Antigen Retrieval:

  • Critical for unmasking epitopes in FFPE tissues

  • Recommended methods for RFTN2:

    • TE buffer pH 9.0 (primary recommendation)

    • Alternatively, citrate buffer pH 6.0

• Antibody Dilution:

  • Typical dilution range: 1:50-1:500 for IHC

  • Optimization for each tissue type is recommended

• Detection Systems:

  • Polymer-based detection systems often provide better signal-to-noise ratio

  • Chromogenic substrates like DAB are commonly used

• Controls:

  • Positive control tissues (e.g., human hippocampus has been verified for some RFTN2 antibodies)

  • Negative controls (primary antibody omission, isotype controls)

  • Blocking with immunizing peptide where available

• Counterstaining:

  • Light hematoxylin counterstaining helps visualize tissue architecture

Researchers should initially test multiple dilutions to determine optimal staining conditions for their specific tissue of interest and RFTN2 antibody.

How Does RFTN2 Expression Vary Across Different Tissues and Cell Types?

Understanding the expression pattern of RFTN2 across tissues is important for experimental design and interpretation:

RFTN2 expression has significant association with cell types involved in immune responses, consistent with its role in immune signaling pathways. The Pharos database indicates that the highest knowledge value (0.89 on a 0-1 scale) for RFTN2 is related to cell type or tissue expression patterns .

Researchers investigating RFTN2 should consider these expression patterns when selecting appropriate experimental models and interpreting results in the context of tissue-specific functions.

What Technical Challenges Are Common When Working with RFTN2 Antibodies?

Researchers may encounter several technical challenges when working with RFTN2 antibodies:

• Background Signal Issues:

  • Polyclonal antibodies may show some non-specific binding

  • Optimization of blocking conditions is critical (typically 5% BSA or milk)

  • Increasing washing steps or duration may help reduce background

• Epitope Masking in Fixed Tissues:

  • Fixation can mask epitopes, requiring optimization of antigen retrieval methods

  • Testing both heat-induced epitope retrieval (HIER) with different buffers may be necessary

• Cross-Reactivity with Related Proteins:

  • RFTN2 belongs to the raftlin family; ensure the antibody doesn't cross-react with other family members

  • Validate specificity in systems with known expression profiles

• Variable Performance Across Applications:

  • An antibody that works well for Western blotting may not perform optimally for IHC or IF

  • Application-specific validation is essential

• Batch-to-Batch Variability:

  • Particularly relevant for polyclonal antibodies

  • Consider purchasing larger quantities of a single lot for long-term studies

When troubleshooting, methodically adjust one parameter at a time while keeping detailed records of protocol modifications and observed outcomes.

How Can RFTN2 Antibodies Be Used to Study Its Interaction with Signaling Partners?

RFTN2 interacts with several key signaling proteins, and antibodies can be valuable tools for investigating these interactions:

• Co-Immunoprecipitation (Co-IP):

  • RFTN2 antibodies can be used to pull down RFTN2 along with its binding partners

  • Particularly useful for studying interactions with known partners like LAT and SYK

  • Protocol considerations:

    • Gentle lysis conditions to preserve protein-protein interactions

    • Cross-linking may be necessary for transient interactions

    • Controls should include IgG from the same species as the RFTN2 antibody

• Proximity Ligation Assay (PLA):

  • Provides in situ detection of protein-protein interactions

  • Requires antibodies against both RFTN2 and its potential interaction partner from different host species

• Immunofluorescence Co-localization:

  • RFTN2 antibodies can be used in conjunction with antibodies against potential interaction partners

  • Confocal microscopy allows assessment of spatial co-localization

  • Quantitative co-localization analysis should be performed

• FRET/BRET Approaches:

  • Requires fusion proteins rather than antibodies directly

  • RFTN2 antibodies can validate expression of fusion constructs

• Functional Studies:

  • RFTN2 antibodies can be used to confirm knockdown/knockout efficiency when studying the functional consequences of RFTN2 depletion on signaling pathways

These approaches provide complementary information about RFTN2's role in signaling complexes, particularly in immune cell signaling contexts.

What Are the Considerations for Using RFTN2 Antibodies in Flow Cytometry Applications?

While flow cytometry is not explicitly mentioned in the search results as a validated application for RFTN2 antibodies, researchers interested in adapting them for this purpose should consider:

• Cellular Localization Challenges:

  • RFTN2 is primarily a membrane-associated protein involved in lipid rafts

  • Proper permeabilization is crucial for accessing intracellular epitopes

  • Consider using gentle permeabilization methods that preserve membrane structures

• Antibody Selection:

  • Fluorochrome-conjugated antibodies (e.g., FITC-conjugated) are available for RFTN2

  • For unlabeled primary antibodies, select compatible secondary antibodies with appropriate fluorochromes

• Validation Steps:

  • Positive controls: cells known to express RFTN2

  • Negative controls: isotype controls, unstained cells, and secondary-only controls

  • Blocking with immunizing peptide where available

• Protocol Optimization:

  • Titrate antibody concentrations

  • Optimize fixation and permeabilization conditions

  • Consider cell surface marker co-staining for population identification

• Data Analysis Considerations:

  • Establish clear gating strategies

  • Consider median fluorescence intensity (MFI) rather than percent positive for quantification

  • Account for autofluorescence, especially in certain cell types

Given that flow cytometry applications are not specifically validated for most RFTN2 antibodies in the search results, thorough validation would be necessary before using them for critical experiments.

How Can RFTN2 Antibodies Be Used to Study Its Role in B-cell Receptor Signaling?

RFTN2 is implicated in B-cell antigen receptor (BCR) signaling , and antibodies can be powerful tools to investigate this function:

• Biochemical Approaches:

  • Western blotting with RFTN2 antibodies can detect changes in RFTN2 expression or post-translational modifications following BCR stimulation

  • Immunoprecipitation can identify dynamic interaction partners in resting versus activated B cells

• Microscopy-Based Methods:

  • Immunofluorescence with RFTN2 antibodies can track changes in subcellular localization during BCR engagement

  • Co-localization with BCR components and downstream signaling molecules

  • Super-resolution microscopy can provide insights into RFTN2's role in the nanoscale organization of signaling complexes

• Functional Studies:

  • RFTN2 antibodies can validate knockdown efficiency in studies examining the functional consequences of RFTN2 depletion on BCR signaling

  • Phospho-specific antibodies against BCR signaling components can assess signaling outcomes

• Membrane Microdomain Analysis:

  • Given RFTN2's role in lipid rafts, antibodies can be used in detergent-resistant membrane fraction analysis

  • Cholesterol depletion experiments can assess RFTN2's raft-dependent functions

• Calcium Flux Assays:

  • RFTN2 antibodies can validate experimental manipulations of RFTN2 in studies measuring calcium flux following BCR stimulation

These approaches can provide mechanistic insights into how RFTN2 contributes to BCR-mediated B cell activation, potentially identifying targets for immunomodulatory interventions.

What Are the Most Effective Fixation Methods When Using RFTN2 Antibodies for Immunofluorescence?

For optimal immunofluorescence results with RFTN2 antibodies, fixation method selection is critical:

• Paraformaldehyde Fixation:

  • 4% PFA is commonly used for RFTN2 immunofluorescence

  • Preserves cellular morphology while maintaining antigen accessibility

  • Recommended fixation time: 10-15 minutes at room temperature

• Methanol Fixation:

  • May be suitable for certain RFTN2 epitopes

  • Better for preserving certain cytoskeletal elements

  • Protocol: Ice-cold methanol for 5-10 minutes at -20°C

• Hybrid Protocols:

  • PFA followed by methanol can combine benefits of both approaches

  • Particularly useful when co-staining for proteins requiring different fixation methods

• Glutaraldehyde Considerations:

  • Generally not recommended for RFTN2 immunofluorescence due to high autofluorescence

  • If necessary, use at low concentrations (0.1-0.5%) and quench with sodium borohydride

• Permeabilization:

  • Critical step after fixation

  • Options include:

    • 0.1-0.5% Triton X-100 (10 minutes)

    • 0.1-0.5% Saponin (gentler, may better preserve membrane structures)

    • 0.1% Tween-20 (very gentle)

• Optimization Strategy:

  • Test multiple fixatives with your specific RFTN2 antibody

  • Consider co-staining requirements when selecting fixation method

  • Document detailed protocols for reproducibility

Since RFTN2 is associated with membrane microdomains, fixation methods that preserve membrane structures while allowing antibody accessibility to epitopes will likely yield the best results.

How Can Researchers Troubleshoot Inconsistent Results with RFTN2 Antibodies?

When facing inconsistent results with RFTN2 antibodies, systematic troubleshooting approaches can help identify and resolve issues:

• Antibody-Related Factors:

  • Check antibody age, storage conditions, and freeze-thaw cycles

  • Verify working concentration through titration experiments

  • Consider testing antibodies from different suppliers or targeting different epitopes

  • For polyclonal antibodies, batch-to-batch variation can be significant

• Sample Preparation Issues:

  • Ensure consistent protein extraction methods for Western blotting

  • Standardize fixation times and conditions for IHC/IF

  • Verify tissue quality and processing methods

• Technical Parameters:

  • For Western blots:

    • Optimize transfer conditions (time, voltage, buffer composition)

    • Test different blocking agents (BSA vs. milk)

    • Adjust primary antibody incubation (time, temperature)

  • For IHC/IF:

    • Optimize antigen retrieval methods

    • Test different detection systems

    • Adjust antibody dilution and incubation conditions

• Experimental Design Considerations:

  • Include appropriate positive and negative controls in each experiment

  • Use standardized protocols with minimal variations

  • Document all experimental conditions meticulously

• Validation Approaches:

  • Confirm findings using alternative methods

  • Consider using genetic approaches (siRNA, CRISPR) to validate antibody specificity

Maintaining a detailed laboratory notebook with exact protocols and observations is essential for identifying patterns in inconsistent results and developing effective solutions.

What Are Advanced Applications of RFTN2 Antibodies in Studying Innate Immune Signaling?

RFTN2 has been implicated in innate immune signaling, particularly in TLR4-mediated pathways . Advanced applications of RFTN2 antibodies in this field include:

• Investigating TLR4 Internalization Dynamics:

  • RFTN2 mediates clathrin-dependent internalization of TLR4 in dendritic cells

  • Immunofluorescence time-course experiments with RFTN2 antibodies can track co-localization with TLR4 during LPS stimulation

  • Super-resolution microscopy can provide nanoscale insights into internalization complex formation

• Dissecting TICAM1-Mediated Signaling:

  • RFTN2 activates TICAM1-mediated signaling leading to IFNB1 production

  • Biochemical approaches with RFTN2 antibodies can identify interaction partners in this pathway

  • Proximal ligation assays can confirm in situ interactions

• Lipid Raft Microdomain Analysis:

  • As a raft-linking protein, RFTN2 is crucial for microdomain organization

  • Detergent-resistant membrane fractionation followed by Western blotting with RFTN2 antibodies

  • Live-cell imaging with fluorescently-labeled RFTN2 antibody fragments

• Cell-Type Specific Signaling Mechanisms:

  • Compare RFTN2 expression and localization across different innate immune cell types

  • Correlate with functional responses to TLR ligands

• Cross-Talk with Adaptive Immune Signaling:

  • RFTN2's dual role in innate (TLR4) and adaptive (BCR) immunity suggests integrative functions

  • RFTN2 antibodies can help map shared signaling components and pathways