MSRB2 Antibody, FITC conjugated

What is MSRB2 and what is its biological significance?

MSRB2 (Methionine Sulfoxide Reductase B2) is a 182-amino acid protein with an N-terminal mitochondrial targeting signal, a catalytic cysteine, and two zinc-coordinating CxxC motifs. It functions as part of the mitochondrial MSR system, which catalyzes the stereospecific reduction of methionine-R-sulfoxides back to methionine . This enzymatic activity plays a crucial role in cellular defense against oxidative stress by eliminating reactive oxygen species (ROS). MSRB2 specifically protects mitochondrial integrity and enhances cell survival through its ROS scavenging capabilities . Recent research has also identified MSRB2 as a binding partner of LG72, suggesting its potential involvement in the regulation of mitochondrial oxidative stress processes related to certain psychiatric disorders .

What applications are supported by MSRB2 antibodies in research settings?

MSRB2 antibodies support multiple research applications depending on their specific characteristics and conjugation status. Common applications include:

• Western Blotting (WB): For detecting MSRB2 protein expression in tissue and cell lysates, typically observed at approximately 19-20 kDa .

• Immunohistochemistry (IHC): For visualizing MSRB2 localization in paraffin-embedded or frozen tissue sections .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of MSRB2 in samples .

• Immunofluorescence (IF): For subcellular localization studies in both cultured cells and tissue sections .

• Flow Cytometry (FACS): Particularly with fluorophore-conjugated antibodies for quantitative single-cell analysis .

• Immunocytochemistry (ICC): For detailed subcellular localization studies .

The recommended dilution ranges vary by application:

These values should be optimized for each experimental system to obtain optimal results .

What is FITC conjugation and why is it valuable for MSRB2 antibody applications?

FITC (Fluorescein Isothiocyanate) conjugation involves the chemical attachment of a fluorescent FITC molecule to an antibody, enabling direct visualization of the antibody-antigen interaction without requiring secondary detection reagents. The conjugation occurs through the reaction of the isothiocyanate group of FITC with primary amino groups (particularly lysine residues) on the antibody . FITC emits green fluorescence (emission ~520 nm) when excited at ~495 nm, making it compatible with standard fluorescence microscopy and flow cytometry instrumentation.

For MSRB2 research, FITC-conjugated antibodies offer several advantages:

• Direct detection without secondary antibodies, reducing background and cross-reactivity issues

• Suitable for multicolor immunofluorescence when studying MSRB2 co-localization with other mitochondrial proteins

• Enables live-cell imaging applications to monitor MSRB2 dynamics

• Facilitates flow cytometric analysis of MSRB2 expression across cell populations

The FITC-conjugated MSRB2 antibodies are particularly valuable for studies examining mitochondrial oxidative stress responses and protein interactions in cellular models .

How should researchers interpret molecular weight observations for MSRB2 in experimental results?

When analyzing MSRB2 detection results, researchers should expect to observe the protein at approximately 19-20 kDa on Western blots, which aligns with its calculated molecular weight of 20 kDa . The full-length MSRB2 protein consists of 182 amino acids, but researchers should note that:

• The N-terminal mitochondrial targeting sequence may be cleaved upon mitochondrial import, potentially resulting in a slightly smaller observed protein size in mitochondrial fractions

• Post-translational modifications may affect migration patterns

• Different antibodies targeting distinct epitopes (e.g., AA 31-130 vs. AA 102-201) may show slight variations in detection efficiency

When unexpected molecular weight bands appear, researchers should consider:

• Potential protein degradation products

• Splice variants

• Post-translational modifications affecting mobility

• Non-specific antibody binding

Validation through knockout/knockdown controls is recommended when characterizing a new MSRB2 antibody in a specific experimental system .

What are the optimal conditions for FITC conjugation to MSRB2 antibodies?

The optimal conditions for FITC conjugation to antibodies, including those against MSRB2, have been experimentally established through comprehensive research. The following parameters yield maximal labeling efficiency:

• Reaction temperature: Room temperature (20-25°C)

• pH: 9.5 (alkaline conditions favor reactivity of antibody amino groups)

• Protein concentration: 25 mg/ml (initial concentration)

• Reaction time: 30-60 minutes (longer incubation risks over-labeling)

• Buffer composition: Typically carbonate or borate buffers that maintain alkaline pH

The process requires a relatively pure IgG preparation, preferably obtained through DEAE Sephadex chromatography, and high-quality FITC reagent . After conjugation, the separation of optimally labeled antibodies from under- and over-labeled proteins can be achieved by gradient DEAE Sephadex chromatography.

The molecular fluorescein/protein (F/P) ratio is critical for optimal performance - too few FITC molecules result in weak signal, while too many can compromise antibody binding affinity and increase non-specific background. For MSRB2 antibodies, maintaining an F/P ratio of approximately 3-6 FITC molecules per antibody generally provides the best balance of signal intensity and specificity .

What methodological approaches are recommended for studying MSRB2 interactions with binding partners?

When investigating MSRB2 interactions with binding partners such as LG72, researchers should employ multiple complementary techniques to ensure robust findings:

• Co-immunoprecipitation (Co-IP):

  • Utilize MSRB2 antibodies (either FITC-conjugated or unconjugated) to pull down protein complexes from cell lysates

  • Include appropriate controls: IgG control, lysate input control, and reciprocal IP

  • Perform under conditions that preserve physiological interactions (mild detergents, physiological salt concentrations)

• Proximity Ligation Assay (PLA):

  • Combines FITC-conjugated MSRB2 antibody with antibodies against potential binding partners

  • Generates fluorescent signals only when proteins are within 30-40 nm proximity

  • Particularly useful for visualizing interactions in their subcellular context

• Fluorescence Resonance Energy Transfer (FRET):

  • Pair FITC-conjugated MSRB2 antibody with appropriate acceptor fluorophore-conjugated antibodies

  • Enables detection of protein-protein interactions at 1-10 nm resolution

  • Can be analyzed by confocal microscopy or flow cytometry

• Subcellular fractionation:

  • Isolate mitochondrial fractions to enrich for MSRB2-containing complexes

  • Verify purity using markers for different cellular compartments

  • Analyze co-occurrence of potential binding partners using FITC-MSRB2 antibodies in combination with other detection methods

Research examining MSRB2's interaction with LG72 has implicated this protein pair in the regulation of mitochondrial oxidative stress, potentially relevant to psychiatric disorders . This suggests that methodological approaches should include oxidative stress induction paradigms when studying these interactions.

How can researchers optimize protocols for detecting MSRB2 in mitochondria using FITC-conjugated antibodies?

Optimizing mitochondrial MSRB2 detection requires careful consideration of sample preparation, fixation, permeabilization, and imaging parameters:

• Sample preparation:

  • For cell cultures: Grow cells on glass coverslips or in glass-bottom dishes compatible with high-resolution microscopy

  • For tissue sections: Use freshly frozen or properly fixed samples with minimal autofluorescence

• Fixation protocol:

  • Use 4% paraformaldehyde for 10-15 minutes at room temperature

  • Avoid methanol fixation which can extract mitochondrial lipids and disrupt mitochondrial morphology

  • For enhanced mitochondrial structure preservation, consider adding 0.1-0.2% glutaraldehyde

• Permeabilization optimization:

  • Use 0.1-0.2% Triton X-100 for 5-10 minutes

  • For selective plasma membrane permeabilization while preserving mitochondrial membranes, use digitonin at 10-50 μg/ml

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • For directly conjugated FITC-MSRB2 antibodies, use at experimentally optimized dilutions (typically starting at 1:100-1:400)

  • Include 0.1% BSA in antibody dilution buffer to reduce non-specific binding

• Mitochondrial co-labeling:

  • Co-stain with mitochondrial markers (e.g., TOMM20, COX IV) using spectrally distinct fluorophores

  • Consider MitoTracker dyes for live-cell applications prior to fixation

• Counterstaining and mounting:

  • Use DAPI for nuclear counterstaining

  • Mount with anti-fade reagent optimized for FITC fluorescence preservation

• Imaging considerations:

  • Use confocal microscopy for optimal resolution of mitochondrial structures

  • Apply deconvolution algorithms to enhance signal-to-noise ratio

  • Consider super-resolution techniques for detailed colocalization studies

The MSRB2 signal should localize to mitochondria as indicated by co-labeling with established mitochondrial markers, reflecting its biological role in the mitochondrial methionine sulfoxide reduction system .

What controls are essential when using FITC-conjugated MSRB2 antibodies in flow cytometry experiments?

Flow cytometry experiments with FITC-conjugated MSRB2 antibodies require rigorous controls to ensure reliable and interpretable results:

• Unstained control:

  • Cells processed through all permeabilization and preparation steps but without any antibody

  • Establishes baseline autofluorescence level

• Isotype control:

  • FITC-conjugated antibody of the same isotype (e.g., rabbit IgG) but with irrelevant specificity

  • Controls for non-specific binding due to Fc receptors or hydrophobic interactions

• Single-color controls:

  • When performing multicolor analysis, include samples stained with each fluorophore individually

  • Essential for compensation setup and spectral overlap correction

• Blocking validation control:

  • Pre-incubate FITC-MSRB2 antibody with excess recombinant MSRB2 protein before staining

  • Signal should be significantly reduced if binding is specific

• Positive and negative cell line controls:

  • Use cell lines with confirmed high (positive) and low/none (negative) MSRB2 expression

  • Helps establish detection thresholds and gating strategies

• Fixation/permeabilization controls:

  • Compare different fixation and permeabilization protocols to optimize signal intensity

  • Particularly important since MSRB2 is a mitochondrial protein requiring permeabilization of both plasma and mitochondrial membranes

• Antibody titration:

  • Test multiple antibody concentrations to determine optimal signal-to-noise ratio

  • Plot signal-to-noise ratio versus antibody concentration to identify the optimal dilution

• Biological validation controls:

  • Samples with MSRB2 knockdown/knockout

  • Samples with MSRB2 overexpression

  • These validate antibody specificity within the experimental system

Including these controls allows researchers to confidently attribute observed signals to specific MSRB2 detection rather than technical artifacts or non-specific binding events.

How can researchers quantitatively assess MSRB2 expression using FITC-conjugated antibodies?

Quantitative assessment of MSRB2 expression using FITC-conjugated antibodies can be performed through several complementary approaches:

• Flow cytometry quantification:

  • Measure mean fluorescence intensity (MFI) of FITC signal in gated cell populations

  • Use calibration beads with known quantities of FITC molecules to convert MFI to molecules of equivalent soluble fluorochrome (MESF)

  • Compare results across experimental conditions using fold-change or absolute values

• Quantitative microscopy:

  • Capture images using consistent exposure settings across all samples

  • Perform integrated density measurements of FITC signal within defined regions of interest (ROIs)

  • Subtract background from cell-free areas

  • Normalize to cell number or area as appropriate

• Fluorescence microplate reader analysis:

  • For high-throughput screening applications

  • Plate cells at consistent density

  • Process with FITC-MSRB2 antibody staining

  • Measure total fluorescence and normalize to cell number (using parallel wells with cell counting dye)

• Quantitative Western blotting correlation:

  • Perform parallel analyses using unconjugated MSRB2 antibodies in Western blotting

  • Create standard curves using recombinant MSRB2 protein

  • Correlate Western blot band intensities with fluorescence measurements to validate findings

• Comparative protein profiling:

  • Use FITC-conjugated MSRB2 antibodies in combination with other fluorescently labeled antibodies

  • Apply methodologies like the dilution series shown in the table below for Western blotting applications:

When quantifying MSRB2 expression, researchers should consider that this protein localizes to mitochondria and its expression levels may correlate with mitochondrial content and oxidative stress conditions .

What are common issues when using FITC-conjugated MSRB2 antibodies and how can they be resolved?

Researchers working with FITC-conjugated MSRB2 antibodies may encounter several technical challenges that can be systematically addressed:

• High background fluorescence:

  • Cause: Over-conjugation of FITC, insufficient blocking, or non-specific binding

  • Solution: Optimize blocking conditions (try 5% BSA or 10% normal serum), increase washing steps, use a more dilute antibody solution, or select an antibody with optimal F/P ratio (3-6 FITC molecules per antibody)

• Weak or absent specific signal:

  • Cause: Inadequate permeabilization (especially critical for mitochondrial proteins), insufficient antibody concentration, epitope masking, or protein denaturation

  • Solution: Test different permeabilization agents and times, increase antibody concentration, try alternative antigen retrieval methods, or use antibodies targeting different MSRB2 epitopes

• Photobleaching of FITC signal:

  • Cause: Extended exposure to excitation light

  • Solution: Use anti-fade mounting media, minimize exposure during imaging, employ computational approaches like deconvolution to enhance signal from lower exposures

• Cross-reactivity with non-target proteins:

  • Cause: Antibody binds to proteins with similar epitopes

  • Solution: Validate specificity through knockout/knockdown controls, perform competitive blocking with recombinant MSRB2 protein, or test alternative antibodies targeting different MSRB2 epitopes

• Suboptimal mitochondrial localization:

  • Cause: Incomplete permeabilization of mitochondrial membranes or disruption of mitochondrial morphology

  • Solution: Optimize fixation and permeabilization protocols specifically for mitochondrial preservation, use mitochondrial co-markers to verify proper sample preparation

• Variable results across experiments:

  • Cause: Inconsistent antibody quality, variations in cell density or state, or protocol deviations

  • Solution: Use antibodies from the same lot, standardize cell culture conditions, and strictly adhere to optimized protocols

Maintaining detailed records of troubleshooting experiments and performing systematic parameter optimization is essential for establishing reliable protocols for MSRB2 detection using FITC-conjugated antibodies.

How does the choice of amino acid region targeted by MSRB2 antibodies affect experimental outcomes?

The selection of specific amino acid regions for MSRB2 antibody targeting can significantly impact experimental results and application suitability:

• Epitope accessibility considerations:

  • Antibodies targeting the N-terminal region (AA 10-182, AA 21-182) may have limited accessibility if the mitochondrial targeting sequence is involved in protein-protein interactions or membrane insertion

  • Mid-region epitopes (AA 31-130) are often more accessible in folded proteins and may provide more consistent detection across applications

  • C-terminal epitopes may be preferable for detecting processed forms of MSRB2 after mitochondrial import

• Application-specific performance:

  • Western blotting: Antibodies targeting linear epitopes (such as AA 31-130) typically perform well as proteins are denatured

  • Immunoprecipitation: Antibodies recognizing surface-exposed epitopes in the native conformation are preferred

  • Immunofluorescence: Detection efficiency varies by epitope accessibility within fixed and permeabilized samples

• Cross-reactivity profile:

  • Antibodies targeting highly conserved regions may cross-react with MSRB2 from multiple species (mouse, rat, human)

  • Those targeting less conserved regions may provide species specificity but potentially limited cross-reactivity

• Functional domain considerations:

  • Antibodies targeting catalytic domains (containing the catalytic cysteine or zinc-coordinating CxxC motifs) may interfere with enzymatic activity in functional assays

  • Epitopes near protein interaction sites may block or alter binding to partners like LG72

• Post-translational modification detection:

  • Epitopes containing potential modification sites may show variable detection depending on the modification status

  • Antibodies specifically designed to recognize modified forms may be needed for studying regulatory mechanisms

Researchers should select MSRB2 antibodies based on the specific requirements of their experimental system and application, with consideration of the biological questions being addressed. When possible, validating findings with multiple antibodies targeting different epitopes provides the most robust results.

What emerging methodologies might enhance MSRB2 research using fluorescently conjugated antibodies?

Several cutting-edge approaches show promise for advancing MSRB2 research beyond conventional applications of FITC-conjugated antibodies:

• Super-resolution microscopy techniques:

  • Stimulated emission depletion (STED) microscopy for visualizing precise mitochondrial localization of MSRB2

  • Single-molecule localization microscopy (PALM/STORM) for mapping MSRB2 distribution patterns at nanometer-scale resolution

  • Structured illumination microscopy (SIM) for improved visualization of MSRB2 in relation to mitochondrial substructures

• Live-cell imaging advances:

  • Cell-permeable nanobodies conjugated with FITC for real-time monitoring of MSRB2 dynamics without fixation artifacts

  • Correlative light and electron microscopy (CLEM) combining fluorescence detection with ultrastructural analysis

  • FRET-based biosensors for monitoring MSRB2 activity and conformational changes in living cells

• Single-cell analytical approaches:

  • Imaging flow cytometry combining high-throughput single-cell analysis with spatial information about MSRB2 localization

  • Mass cytometry (CyTOF) using metal-conjugated antibodies for highly multiplexed analysis of MSRB2 in relation to numerous other proteins

  • Single-cell proteomics correlating MSRB2 expression with broader proteomic profiles

• Advanced molecular interaction studies:

  • Bioluminescence resonance energy transfer (BRET) assays for studying MSRB2 interactions with minimal photodamage

  • Protein complementation assays adapted for mitochondrial proteins to study MSRB2 interactions in their native environment

  • Optogenetic approaches to manipulate MSRB2 activity while monitoring cellular responses

• Integration with multi-omics datasets:

  • Combining FITC-MSRB2 antibody-based analyses with transcriptomics, proteomics, and metabolomics

  • Spatial transcriptomics to correlate MSRB2 protein localization with gene expression patterns

  • Systems biology approaches integrating MSRB2 into mitochondrial stress response networks

These methodological advances will enable researchers to address more sophisticated questions about MSRB2's role in mitochondrial redox biology, its regulation in response to oxidative stress, and its potential involvement in pathological conditions related to mitochondrial dysfunction .

What are the methodological approaches for studying MSRB2's role in oxidative stress using immunofluorescence techniques?

Investigating MSRB2's role in oxidative stress using immunofluorescence requires carefully designed experimental protocols that integrate oxidative challenge paradigms with FITC-conjugated antibody detection:

• Oxidative stress induction protocols:

  • Hydrogen peroxide (H₂O₂) treatment: 100-500 μM for acute exposure

  • Paraquat exposure: 10-100 μM for mitochondria-specific oxidative stress

  • Hypoxia-reoxygenation models for physiologically relevant ROS generation

  • Glutathione depletion (BSO treatment) for compromising endogenous antioxidant systems

• Time-course analysis design:

  • Short-term exposures (minutes to hours) to capture immediate responses

  • Extended treatments (days) to assess adaptive mechanisms

  • Recovery periods following oxidative challenge to evaluate system resilience

• Multiplexed detection strategies:

  • Co-staining with oxidative damage markers (8-oxo-dG, 4-HNE, protein carbonyls)

  • Combination with ROS-sensitive dyes (MitoSOX, CellROX) in live-cell imaging prior to fixation

  • Parallel detection of other antioxidant system components

• Quantitative imaging approaches:

  • Intensity correlation analysis between MSRB2-FITC signal and oxidative stress markers

  • Mitochondrial morphology quantification in relation to MSRB2 expression levels

  • Subcellular redistribution analysis following oxidative challenge

• Genetic manipulation coupled with imaging:

  • MSRB2 overexpression models to assess protective capacity

  • siRNA/shRNA knockdown or CRISPR/Cas9 knockout to evaluate loss-of-function effects

  • Site-directed mutagenesis of catalytic residues to create enzymatically inactive controls

• Functional correlation experiments:

  • Combined immunofluorescence with mitochondrial functional assays

  • Sequential analysis of MSRB2 localization followed by mitochondrial membrane potential measurement

  • Correlation of MSRB2 expression patterns with cell viability following oxidative challenge

These methodological approaches enable researchers to address key questions about MSRB2's protective role against oxidative damage, its regulation in response to oxidative stress, and its potential as a therapeutic target in conditions characterized by mitochondrial dysfunction and oxidative damage .