mStrawberry Monoclonal Antibody

What is mStrawberry and why are monoclonal antibodies developed against it?

mStrawberry is a red fluorescent protein variant used extensively in molecular and cellular biology as a fluorescent tag. Monoclonal antibodies against mStrawberry have been developed to detect both the native mStrawberry protein and fusion proteins where mStrawberry is used as a tag. These antibodies enable researchers to track mStrawberry-tagged proteins in applications where direct fluorescence detection may be compromised or insufficient. The antibodies typically recognize specific epitopes within the mStrawberry protein structure, allowing for high-specificity detection in various experimental contexts .

What experimental applications are mStrawberry monoclonal antibodies most suited for?

mStrawberry monoclonal antibodies are primarily optimized for Western blot (WB) applications, making them ideal for protein detection in cell and tissue lysates. They enable researchers to verify expression of mStrawberry fusion proteins in experimental systems where direct fluorescence visualization is challenging . Unlike direct fluorescence detection, antibody-based detection maintains sensitivity even when protein conformation is disrupted during sample processing. This makes these antibodies particularly valuable for confirming protein expression in denatured samples or when fluorescence signal is too weak for direct visualization.

How does the specificity of mStrawberry monoclonal antibodies affect experimental design?

The specificity profile of mStrawberry monoclonal antibodies requires careful experimental planning. While these antibodies primarily detect mStrawberry and mStrawberry tag proteins, they may exhibit cross-reactivity with mRFP protein due to structural similarities . This cross-reactivity should be considered when designing experiments with multiple fluorescent protein variants. Control experiments using cells expressing other red fluorescent proteins should be included to establish antibody specificity in your particular experimental system. Validation with appropriate positive and negative controls is essential before embarking on complex experimental designs.

What is the recommended protocol for using mStrawberry monoclonal antibodies in Western blotting?

For optimal Western blot results with mStrawberry monoclonal antibodies, standard immunoblotting procedures should be followed with specific adaptations. Based on available research protocols, cell lysates should be prepared in RIPA buffer containing protease inhibitors, and proteins separated on 10-12% SDS-PAGE gels. After transfer to PVDF or nitrocellulose membranes, blocking with 5% non-fat milk or 1% BSA in TBST is recommended . The primary mStrawberry monoclonal antibody should be diluted to 1:1000-1:3000 in blocking buffer and incubated overnight at 4°C. Following washing steps, an appropriate species-specific secondary antibody (typically anti-mouse IgG for most commercial mStrawberry antibodies) conjugated to HRP should be applied. Detection sensitivity can be optimized using enhanced chemiluminescence systems.

How should researchers validate the specificity of mStrawberry monoclonal antibodies in their experimental system?

Validation of mStrawberry monoclonal antibodies requires a multi-step approach. First, positive controls using purified mStrawberry protein or lysates from cells transfected with mStrawberry expression vectors should be included alongside experimental samples. Second, negative controls with non-transfected cells or cells expressing different fluorescent proteins should be used to assess cross-reactivity . For advanced validation, competitive assays with synthetic peptides corresponding to the antibody epitope can be performed, similar to methodology used for other monoclonal antibodies . Additionally, correlation between antibody detection and direct fluorescence visualization provides further validation of specificity and performance.

What storage and handling precautions ensure optimal performance of mStrawberry monoclonal antibodies?

To maintain antibody integrity and performance, mStrawberry monoclonal antibodies should be stored at -20°C according to manufacturer specifications . Most commercial preparations are formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide to prevent microbial growth and maintain stability. Repeated freeze-thaw cycles should be avoided as they can lead to antibody degradation and reduced performance. For routine use, small aliquots should be prepared during initial thawing to minimize exposure to temperature fluctuations. Working dilutions should be prepared fresh before each experiment and stored at 4°C for no more than one week.

How can researchers address weak or absent signal when using mStrawberry monoclonal antibodies?

When encountering weak or absent signals with mStrawberry monoclonal antibodies, systematic troubleshooting approaches should be implemented. First, verify protein expression using direct fluorescence microscopy if possible. If fluorescence is detected but antibody signal is weak, optimize antibody concentration by testing a range of dilutions (1:500 to 1:5000). Increasing incubation time or temperature may enhance signal strength. For Western blotting, increasing protein loading amount or using more sensitive detection reagents can improve results. Additionally, some preparation methods may denature epitopes; therefore, testing native conditions or different lysis buffers might preserve antibody recognition sites. Finally, ensure secondary antibody compatibility with the primary antibody isotype (typically IgG1 for many mStrawberry monoclonal antibodies) .

What strategies minimize background when working with mStrawberry monoclonal antibodies in immunoassays?

High background signal can obscure specific detection with mStrawberry monoclonal antibodies. To minimize background, increase blocking stringency by using 5% BSA instead of milk, which can sometimes cause higher background with certain antibodies. Extended blocking times (2-3 hours at room temperature or overnight at 4°C) may also help. In Western blotting, increase wash duration and frequency between antibody incubations using TBST (0.1% Tween-20). Using validated antibody dilutions is crucial, as too concentrated antibody solutions often increase non-specific binding. Implementing more stringent washing procedures similar to those used in ELISA protocols, which involve 5-6 washes with PBST between incubation steps, can significantly reduce background .

How can researchers differentiate between true mStrawberry signal and autofluorescence or cross-reactivity?

Differentiating true mStrawberry antibody signal from artifacts requires robust controls. Include samples from non-transfected cells processed identically to experimental samples to assess baseline signal and potential cross-reactivity with endogenous proteins. For fluorescence-based detection methods, spectral analysis can help distinguish between mStrawberry fluorescence (emission peak ~610 nm) and cellular autofluorescence, which typically has different spectral characteristics. When using immunohistochemistry or immunofluorescence, secondary antibody-only controls help identify non-specific binding of detection reagents. For advanced applications, conducting peptide competition assays where the antibody is pre-incubated with purified mStrawberry protein before application to samples can confirm signal specificity—true mStrawberry signals should be abolished or significantly reduced in these conditions .

How can mStrawberry monoclonal antibodies enhance tracking of therapeutic proteins in CNS research?

In central nervous system (CNS) research, mStrawberry monoclonal antibodies offer powerful tools for tracking protein distribution and biodistribution studies. As demonstrated in recent cancer therapy research, mStrawberry-tagged cells (such as the 2F7-BR44 lymphoma cells) allow quantitative assessment of disease burden in both systemic tissues and the CNS through flow cytometry of dissociated tissues . For therapeutic protein tracking, researchers can develop dual detection systems where the therapeutic protein is tagged with mStrawberry, allowing both direct fluorescence visualization in live imaging and more sensitive antibody-based detection in fixed tissues or biochemical assays. This approach is particularly valuable when studying blood-brain barrier penetration of therapeutic proteins, as exemplified in studies with rituximab (RTX) nanocapsules, where quantification in brain tissue required sensitive detection methods .

What approaches enable quantitative analysis using mStrawberry monoclonal antibodies in complex tissue samples?

Quantitative analysis in complex tissue samples requires optimized protocols combining tissue processing and antibody-based detection. For brain tissue samples, perfusion with cold PBS prior to tissue collection removes blood contamination that could confound results . Tissue homogenization in the presence of protease inhibitors preserves protein integrity. For quantification, ELISA-based detection offers high sensitivity, with detection limits approaching those of other well-optimized immunoassays (~0.01 μg/L) . Standard curves using purified mStrawberry protein enable accurate quantification. When analyzing tissues with potential interfering substances, sample pretreatment with acetate buffer (pH 5.4) may enhance detection sensitivity, similar to methods used for other therapeutic antibodies in tissue homogenates .

How can mStrawberry monoclonal antibodies be integrated with other detection systems for multiplexed analysis?

Multiplexed analysis incorporating mStrawberry monoclonal antibodies requires strategic planning of detection channels and antibody combinations. For flow cytometry applications, mStrawberry's fluorescence properties (excitation ~574 nm, emission ~596 nm) allow simultaneous detection with fluorophores in the violet, blue, and far-red channels. When designing multiplexed immunoassays, mStrawberry monoclonal antibodies can be paired with antibodies raised in different host species (e.g., rabbit) to enable discrimination using species-specific secondary antibodies. For advanced applications, direct conjugation of mStrawberry monoclonal antibodies to biotin, HRP, or fluorophores with non-overlapping spectra allows simultaneous detection of multiple targets. In microscopy applications, the combination of direct mStrawberry fluorescence with antibody-based detection of other proteins enables correlation between tagged protein localization and interacting partners or cellular structures .

How does epitope selection in mStrawberry monoclonal antibodies affect experimental applications?

Epitope selection is crucial for mStrawberry monoclonal antibody function across different applications. Commercial antibodies are typically raised against synthetic peptides derived from the mStrawberry sequence . When mStrawberry is used as a fusion tag, the epitope accessibility may be affected by the fusion partner's structure or orientation. Some antibodies may recognize linear epitopes (denaturation-resistant), making them ideal for Western blotting but potentially less effective for immunoprecipitation of native proteins. Others may target conformational epitopes, optimal for detecting properly folded proteins in non-denaturing applications. When selecting antibodies for specific applications, researchers should consider whether epitopes are located at termini or within internal sequences, as terminal epitopes may be masked in certain fusion constructs.

What methodologies can researchers use to map epitopes recognized by their mStrawberry monoclonal antibodies?

Epitope mapping provides valuable information for optimizing antibody applications. For mStrawberry monoclonal antibodies, peptide array analysis using synthesized overlapping peptides (typically 12-20 amino acids) covering the entire mStrawberry sequence can identify linear epitopes . For this approach, peptides are synthesized on cellulose membranes using Fmoc chemistry with a peptide synthesizer. After synthesis, membranes are probed with the antibody of interest followed by appropriate secondary antibody detection. Competitive ELISA represents another valuable approach, where synthesized peptides compete with immobilized mStrawberry protein for antibody binding. Decreasing signal indicates the peptide contains the epitope. For conformational epitopes, more sophisticated approaches involving hydrogen-deuterium exchange mass spectrometry or X-ray crystallography of antibody-antigen complexes may be required .

How can researchers select the optimal mStrawberry monoclonal antibody clone for their specific application?

Selection of the optimal monoclonal antibody clone should be guided by application requirements and comprehensive validation. For detection of mStrawberry in fixed tissues, antibodies recognizing linear epitopes offer advantages as fixation often denatures proteins. For co-immunoprecipitation studies, antibodies targeting surface-exposed epitopes that don't interfere with protein-protein interactions are preferred. When multiple antibody clones are available (such as the "Mix" formulation mentioned in the search results), testing individual clones for specific applications may yield superior results for particular experiments . For quantitative applications like ELISA, antibodies with documented affinity constants provide more reliable quantification. Ultimately, validation in your specific experimental system remains essential, as performance can vary with expression levels, fusion partners, and experimental conditions.

What emerging technologies might enhance the utility of mStrawberry monoclonal antibodies in research?

Emerging technologies promise to expand mStrawberry monoclonal antibody applications. Nanotechnology approaches, similar to those described for therapeutic antibodies, could enhance detection sensitivity in complex samples. The encapsulation methods used for rituximab that improved CNS delivery could be adapted for mStrawberry antibodies to enhance tissue penetration in thick specimens . Super-resolution microscopy techniques combined with mStrawberry antibody detection could enable visualization of tagged proteins below the diffraction limit. Automation of epitope mapping using peptide array technology may streamline antibody characterization . Furthermore, recombinant antibody engineering could yield mStrawberry-specific single-chain antibodies or nanobodies with superior tissue penetration and reduced background for in vivo applications.