MCF2 Antibody

What is MCF2 and what types of antibodies are available for its detection?

MCF2 (MCF.2 cell line derived transforming sequence) has multiple synonyms including ARHGEF22 and KIAA0861 . Several antibody options are available for MCF2 detection, primarily including:

• Polyclonal antibodies from rabbit hosts that react with human MCF2

• Antibodies validated for various applications including ELISA, IHC, IF, WB, and FACS

• Products with different conjugation states (unconjugated or with specific fluorophores)

The selection of an appropriate antibody depends on your specific experimental needs and the techniques you plan to employ. Most commercially available MCF2 antibodies are affinity-isolated and tested against standardized protein arrays to ensure specificity .

How do polyclonal and monoclonal MCF2 antibodies differ in research applications?

The distinction between polyclonal and monoclonal antibodies has significant implications for MCF2 research:

Polyclonal MCF2 antibodies:

• Recognize multiple epitopes on the MCF2 protein

• Provide stronger signal due to multiple binding sites

• More resistant to sample preparation variations

• Typically derived from rabbit hosts as seen in product catalogs

• Useful when protein abundance is low or detection sensitivity is critical

Monoclonal antibodies:

• Recognize a single epitope with high specificity

• Offer greater consistency between lots

• Ideal for distinguishing between closely related proteins

• Better suited for quantitative applications

• Recommended when background interference is problematic

The choice between polyclonal (like product code ABIN7166480) and monoclonal options should be guided by your specific experimental requirements .

What criteria should guide MCF2 antibody selection for specific research applications?

When selecting an MCF2 antibody, consider:

• Application compatibility: Verify validation for your intended technique (WB, IHC, IF, ELISA, FACS)

• Species reactivity: Ensure compatibility with your experimental model (human-reactive antibodies are most common)

• Validation depth: Premium antibodies like Prestige Antibodies undergo extensive validation:

  • Testing on tissue arrays of 44 normal human tissues and 20 cancer types

  • Screening against protein arrays of 364 human recombinant proteins

• Immunogen sequence: Understanding the target region helps predict cross-reactivity and epitope accessibility. For example, MCF2L2 antibody HPA038947 targets a specific amino acid sequence that influences its recognition properties

• Working concentration guidelines: For HPA038947, recommended dilutions are:

  • Immunofluorescence: 0.25-2 μg/mL

  • Immunohistochemistry: 1:50-1:200

Why is the immunogen sequence important when selecting an MCF2 antibody?

The immunogen sequence determines what portion of the MCF2 protein an antibody recognizes. For instance, antibody HPA038947 targets the sequence: "PHPESSPKWVSSKTSQPSTSVPLARPLRTSEEPYTETELNSRGKEDDETKFEVKSEEIFESHHERGNPELEQQARLGDLSPRRRIIRD" .

This information is critical because:

• It helps predict potential cross-reactivity with structurally similar proteins

• It determines whether splice variants or isoforms will be recognized

• It indicates whether post-translational modification sites might affect binding

• It suggests whether the epitope will be accessible in different experimental contexts (native vs. denatured)

• It assists in interpreting unexpected results or discrepancies between different detection methods

What validation methods should be employed to confirm MCF2 antibody specificity?

Rigorous validation ensures reliable experimental outcomes with MCF2 antibodies:

• Knockout/knockdown controls: Test antibody in samples with reduced MCF2 expression

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to block specific binding

• Multi-antibody approach: Compare results from antibodies targeting different MCF2 epitopes

• Cross-reactivity assessment: Test on tissues known to express structurally similar proteins

• Protein array screening: Similar to validation performed on Prestige Antibodies across 364 human recombinant proteins

• Tissue panel verification: Test across positive and negative tissue controls, following the approach used in Human Protein Atlas validation

• Immunoprecipitation-mass spectrometry: Confirm identity of captured proteins

Implementing multiple validation strategies provides the strongest evidence for antibody specificity and increases confidence in experimental results.

How should researchers determine optimal working concentrations for MCF2 antibodies?

Determining the optimal working concentration involves systematic optimization:

• Begin with manufacturer recommendations: For example, HPA038947 suggests:

  • Immunofluorescence: 0.25-2 μg/mL

  • Immunohistochemistry: 1:50-1:200 dilution

• Titration experiment protocol:

  • Test 5-7 concentrations in 2-fold serial dilutions around the recommended range

  • Include positive control (tissue known to express MCF2) and negative control (tissue without MCF2 expression)

  • Evaluate signal-to-noise ratio rather than absolute signal intensity

  • Select concentration that maximizes specific signal while minimizing background

• Application-specific considerations:

  • Western blot: Lower concentrations typically needed compared to IHC

  • Flow cytometry: May require higher concentrations than IF

  • Adjust based on fixation methods and sample types

What controls are essential when using MCF2 antibodies in experimental workflows?

Proper controls are critical for meaningful MCF2 antibody experiments:

• Positive controls:

  • Tissues/cells with confirmed MCF2 expression

  • Recombinant MCF2 protein

  • Transfected cells overexpressing MCF2

• Negative controls:

  • Tissues known to lack MCF2 expression

  • MCF2 knockout/knockdown samples

  • Technical negative (primary antibody omission)

  • Isotype controls (especially for flow cytometry)

• Validation controls:

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

  • Blocking peptide competition to confirm specificity

  • Multiple antibodies targeting different MCF2 epitopes

• Method-specific controls:

  • For IHC/IF: Autofluorescence control

  • For Western blot: Loading control and molecular weight marker

  • For IP experiments: IgG control pull-down

The Human Protein Atlas project exemplifies thorough control implementation with extensive tissue arrays and protein cross-reactivity testing for antibodies like HPA038947 .

What are the optimal protocols for using MCF2 antibodies in immunohistochemistry?

For successful MCF2 immunohistochemistry:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin (24 hours)

  • Process and embed in paraffin

  • Section at 4-5 μm thickness on positively charged slides

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval methods:
    a) Citrate buffer (pH 6.0)
    b) EDTA buffer (pH 9.0)

  • Heat to 95-98°C for 20 minutes

  • Allow gradual cooling to room temperature

• Blocking and antibody application:

  • Block endogenous peroxidase (3% H₂O₂, 10 minutes)

  • Block non-specific binding (5% normal serum, 1 hour)

  • Apply MCF2 antibody at optimized dilution (starting with 1:50-1:200 for HPA038947)

  • Incubate overnight at 4°C in a humidified chamber

• Detection and visualization:

  • Apply appropriate HRP-conjugated secondary antibody

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount

• Critical controls:

  • Include tissue with known MCF2 expression

  • Include negative control section (primary antibody omitted)

  • Document all staining patterns with photomicrographs

How should researchers optimize Western blotting protocols for MCF2 detection?

For optimal Western blot detection of MCF2:

• Sample preparation considerations:

  • Extract proteins using RIPA buffer with protease inhibitors

  • Determine protein concentration by BCA or Bradford assay

  • Load 25-50 μg total protein per lane

  • Denature samples (95°C for 5 minutes) in reducing Laemmli buffer

• Gel electrophoresis and transfer parameters:

  • Separate on 8-10% SDS-PAGE (MCF2 is a relatively large protein)

  • Transfer to PVDF membrane (better protein retention than nitrocellulose)

  • Use wet transfer system (30V overnight at 4°C for large proteins)

  • Verify transfer efficiency with reversible protein stain

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST (1 hour, room temperature)

  • Incubate with primary antibody at optimized dilution in 5% BSA/TBST

  • Incubate overnight at 4°C with gentle agitation

  • Wash extensively (4 × 10 minutes with TBST)

  • Apply HRP-conjugated secondary antibody (1:5000, 1 hour)

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) substrate

  • Begin with short exposures (30 seconds) and increase as needed

  • Consider using signal enhancers for low-abundance targets

• Troubleshooting strategies:

  • If high background: Increase blocking, reduce antibody concentration

  • If weak signal: Increase protein load, longer exposure, enhanced ECL

  • If multiple bands: Validate with knockout controls, consider isoforms

What are the critical considerations for MCF2 detection using immunofluorescence?

For successful immunofluorescence detection of MCF2:

• Fixation optimization:

  • Test both 4% paraformaldehyde (10 minutes) and methanol (-20°C, 10 minutes)

  • Different fixatives may preserve different epitopes

  • Document fixation impact on signal intensity and pattern

• Permeabilization strategies:

  • For paraformaldehyde-fixed samples: 0.1-0.5% Triton X-100 (10 minutes)

  • For methanol-fixed samples: Additional permeabilization usually unnecessary

  • Optimize concentration and time based on cell type

• Antibody incubation parameters:

  • Block with 5% normal serum from secondary antibody host species

  • Apply MCF2 antibody at optimized concentration (0.25-2 μg/mL for HPA038947)

  • Incubate overnight at 4°C in humidified chamber

  • Wash extensively (4 × 5 minutes with PBS)

• Signal detection and imaging:

  • Use appropriate fluorophore-conjugated secondary antibody

  • Include DAPI nuclear counterstain

  • Mount with anti-fade mounting medium

  • Capture z-stack images for co-localization studies

  • Use consistent exposure settings across experimental conditions

• Critical controls:

  • Primary antibody omission control

  • Cells with varied MCF2 expression levels

  • Competitive blocking with immunizing peptide

How should flow cytometry protocols be adapted for MCF2 detection?

For flow cytometric analysis of MCF2:

• Cell preparation considerations:

  • Harvest adherent cells using enzyme-free dissociation buffers

  • Fix with 2-4% paraformaldehyde (10 minutes)

  • Permeabilize with 0.1% saponin-containing buffer for intracellular MCF2

  • Maintain samples at 4°C throughout processing

• Staining protocol optimization:

  • Block with 2% FBS and 1% BSA in PBS (30 minutes)

  • If using immune cells, include Fc receptor blocking step

  • Apply MCF2 antibody at predetermined concentration

  • Include viability dye to exclude dead cells

  • Perform all washes in buffer containing permeabilization agent

• Essential controls:

  • Unstained cells for autofluorescence assessment

  • Isotype control at matching concentration

  • Fluorescence-minus-one (FMO) controls

  • Positive and negative cell lines

• Instrument setup and analysis:

  • Optimize PMT voltages using unstained and single-stained controls

  • Perform compensation when using multiple fluorophores

  • Establish gating strategy based on controls

  • Collect sufficient events (minimum 10,000 in target population)

• Data presentation:

  • Report both percentage positive and median fluorescence intensity

  • Use consistent gating and scales across experimental groups

  • Include representative histogram overlays showing shifts in MCF2 expression

How should researchers troubleshoot weak or absent signal with MCF2 antibodies?

When facing weak or absent MCF2 signal:

• Antibody-related considerations:

  • Verify antibody activity with dot blot of recombinant MCF2

  • Check storage conditions and expiration date

  • Increase concentration (2-5 fold above recommended)

  • Try alternative MCF2 antibody targeting different epitope

• Sample-related factors:

  • Confirm MCF2 expression in your samples (mRNA level)

  • Assess protein degradation with fresh samples

  • For IHC/IF: Optimize antigen retrieval conditions

  • For WB: Test different extraction methods (RIPA vs. NP-40)

• Technical optimization:

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

  • For WB: Use PVDF instead of nitrocellulose membranes

  • For IHC/IF: Test signal amplification systems

  • For WB: Increase protein loading (50-100 μg)

• Systematic approach:

  • Begin with positive control (known MCF2-expressing sample)

  • Isolate variables by changing one parameter at a time

  • Document all modifications to protocol

  • Consider epitope accessibility issues (try alternative sample preparation)

What strategies should be employed when facing high background or non-specific binding?

To address high background with MCF2 antibodies:

• Antibody optimization:

  • Titrate to lower concentration

  • Increase washing duration and frequency

  • Change diluent (try 1% BSA vs. 5% normal serum)

  • Consider pre-adsorption against irrelevant tissues

• Blocking improvements:

  • Extend blocking time (2-3 hours)

  • Test alternative blocking agents (BSA, normal serum, commercial blockers)

  • Add 0.1-0.3% Triton X-100 to blocking buffer

  • For tissues with high endogenous biotin, add avidin/biotin blocking step

• Sample-specific considerations:

  • Block endogenous peroxidase (3% H₂O₂, 10 minutes) for IHC

  • For tissues with high autofluorescence, use Sudan Black B treatment

  • For WB, add 0.05% SDS to wash buffer for stringent washing

  • For IHC, consider mouse-on-mouse blocking for mouse tissues

• Technical modifications:

  • Reduce incubation temperature (4°C overnight instead of room temperature)

  • Use fresh reagents, particularly detection substrates

  • Filter all solutions to remove particulates

  • For IHC/IF, reduce secondary antibody concentration

How should researchers interpret conflicting results from different MCF2 antibodies?

When different MCF2 antibodies yield conflicting results:

• Epitope-related considerations:

  • Map epitopes of each antibody (consult immunogen sequences)

  • Determine if epitopes are affected by different sample preparation methods

  • Check if epitopes are conserved across species/isoforms being studied

  • Assess if post-translational modifications might mask certain epitopes

• Validation assessment:

  • Review validation data for each antibody

  • Check published literature for known issues

  • Perform your own validation using knockout/knockdown controls

  • Test antibodies on recombinant MCF2 variants

• Methodological investigation:

  • Compare native vs. denatured conditions

  • Test multiple fixation protocols for IF/IHC

  • For WB, compare reducing vs. non-reducing conditions

  • Try different extraction methods to ensure complete protein solubilization

• Resolution approach:

  • Use orthogonal techniques (mRNA analysis, mass spectrometry)

  • Employ genetic approaches (tagged constructs, CRISPR knockout)

  • Consider co-immunoprecipitation with different antibodies

  • Report all findings transparently, acknowledging limitations

• Data interpretation framework:

  • Higher confidence in results confirmed by multiple antibodies

  • When discrepant, favor results from better-validated antibodies

  • Consider biological explanations (isoforms, post-translational modifications)

  • Document all conditions that produce different results

How can computational approaches enhance MCF2 antibody design and selection?

Computational methods offer powerful approaches to antibody research:

• Epitope prediction and antibody design:

  • The AbDesign algorithm leverages information on backbone conformations and sequence-conservation patterns to design antibodies with desired properties

  • This approach segments antibody structures and recombines them in silico to create novel binding molecules

  • Computational design can identify optimal MCF2 epitopes that are surface-exposed and unique

• Structural considerations in design:

  • Preservation of stabilizing interactions between the framework and complementarity-determining regions (CDRs) is critical for antibody stability

  • Joint optimization of antibody stability and binding energy improves outcomes

  • Through iterative design cycles, computational approaches can address nonideal features like long unstructured loops and buried polar networks

• Practical applications in MCF2 research:

  • Predicting cross-reactivity with related proteins

  • Designing antibodies against challenging MCF2 epitopes

  • Optimizing antibody properties (stability, solubility, specificity)

  • Generating antibodies against conserved epitopes across species

• Integration with experimental validation:

  • Computational predictions require experimental validation

  • Five consecutive design/experiment cycles were needed to develop successful computational antibody design approaches

  • Combined computational-experimental approaches offer the strongest path to novel antibody development

What approaches are recommended for studying MCF2 protein-protein interactions?

To study MCF2 protein interactions effectively:

• Co-immunoprecipitation strategies:

  • Use MCF2 antibodies for pull-down of interacting proteins

  • Optimize lysis conditions to preserve relevant interactions

  • Use chemical crosslinking to capture transient interactions

  • Consider epitope accessibility in protein complexes

• Proximity-based methods:

  • BioID approach: MCF2 fusion with promiscuous biotin ligase

  • APEX2 proximity labeling for subcellular interaction mapping

  • Proximity ligation assay (PLA) using MCF2 antibodies and antibodies against potential interactors

  • These methods capture both stable and transient interactions in native cellular contexts

• Fluorescence-based techniques:

  • Förster resonance energy transfer (FRET) for direct interaction monitoring

  • Bimolecular fluorescence complementation (BiFC) for visualization of interaction sites

  • Fluorescence correlation spectroscopy (FCS) for interaction kinetics

  • Live-cell imaging to track dynamic interactions

• Mass spectrometry approaches:

  • Immunoprecipitation followed by mass spectrometry identification

  • SILAC or TMT labeling for quantitative interaction comparison

  • Crosslinking mass spectrometry for interaction interface mapping

  • Multi-dimensional protein identification technology (MudPIT) for complex samples

• Validation frameworks:

  • Reciprocal co-immunoprecipitation to confirm interactions

  • Domain mapping to identify specific interaction regions

  • Functional assays to determine biological relevance

  • Competitive binding experiments to assess interaction specificity

How do post-translational modifications affect MCF2 antibody recognition?

Post-translational modifications (PTMs) significantly impact antibody recognition:

• Common PTMs affecting antibody binding:

  • Phosphorylation: Adds negative charge, potentially disrupting antibody binding

  • Glycosylation: Large modifications can sterically hinder epitope access

  • Ubiquitination: May mask epitopes or alter protein conformation

  • Proteolytic processing: Can remove epitopes entirely

• Experimental strategies to address PTM interference:

  • Use multiple antibodies targeting different regions

  • Employ modification-specific antibodies when available

  • Compare native vs. denatured detection methods

  • Pre-treat samples to remove specific modifications:

    • Phosphatase treatment for phosphorylation

    • PNGase F for N-linked glycosylation

    • Deubiquitinating enzymes for ubiquitination

• Methodological approaches:

  • Two-dimensional gel electrophoresis to separate modified forms

  • Phospho-tag gels to resolve phosphorylated variants

  • Immunoprecipitation followed by PTM-specific Western blotting

  • Mass spectrometry to identify and map modifications

• Interpretation considerations:

  • Signal differences across sample types may reflect PTM changes

  • Subcellular localization differences may be PTM-dependent

  • Treatment-induced changes may alter epitope accessibility

  • Document all conditions affecting antibody recognition

What considerations are essential for multiplexed detection involving MCF2 antibodies?

For successful multiplexed detection with MCF2 antibodies:

• Antibody selection criteria:

  • Verify compatibility of primary antibodies (different host species)

  • Ensure non-overlapping detection systems

  • Test for cross-reactivity between detection systems

  • Validate each antibody individually before multiplexing

• Multiplex immunofluorescence optimization:

  • Select fluorophores with minimal spectral overlap

  • Include single-color controls for compensation

  • Consider sequential staining for closely related targets

  • Use nuclear counterstain for cell identification

  • Employ spectral unmixing for overlapping fluorophores

• Multiplex immunohistochemistry approaches:

  • Traditional: Different chromogens on sequential sections

  • Advanced: Sequential staining with stripping or bleaching

  • Automated multiplex platforms (Vectra, InSituPlex)

  • Tyramide signal amplification for enhanced sensitivity

• Mass cytometry considerations:

  • Metal-conjugated antibodies eliminate spectral overlap

  • Enables simultaneous detection of 40+ targets

  • Requires specialized equipment (CyTOF)

  • Antibody conjugation and validation critical

• Data analysis frameworks:

  • Colocalization analysis for multiple fluorescent signals

  • Single-cell analysis in heterogeneous populations

  • Machine learning approaches for pattern recognition

  • Quantitative assessment of staining intensity and distribution