mCherry Monoclonal Antibody

What is mCherry and why is it used as a protein tag in research?

mCherry is a monomeric red fluorescent protein (mRFP) belonging to the mFruits family, derived from DsRed of Discosoma sea anemones. Unlike green fluorescent proteins (GFPs) which originate from Aequorea victoria jellyfish, mCherry represents a distinct evolutionary lineage of fluorescent proteins . Researchers utilize mCherry as a fusion tag due to its superior properties: improved brightness, exceptional photostability, and extremely rapid maturation rate compared to first-generation red fluorescent proteins .

The protein consists of 236 amino acid residues with a molecular weight of approximately 27-30 kDa when expressed alone . mCherry's structural stability and monomeric nature make it ideal for creating fusion proteins without causing aggregation issues that plague many other fluorescent tags. These properties collectively make mCherry one of the preferred fluorescent proteins for monitoring physiological processes and detecting transgenic expression in live cell imaging and fixed specimen analysis .

How do mCherry monoclonal antibodies function in experimental systems?

mCherry monoclonal antibodies function by specifically binding to epitopes on the mCherry protein structure, enabling detection of mCherry-tagged proteins in various experimental contexts. These antibodies can recognize both native and denatured forms of mCherry or mCherry fusion proteins . The binding specificity is achieved through precise antibody development, typically using recombinant Discosoma sp. mCherry protein as the immunogen .

The antibody-antigen interaction forms the basis for diverse detection methods. In western blotting, the antibodies bind denatured mCherry proteins separated by electrophoresis, enabling visualization through secondary detection systems . In immunocytochemistry and immunohistochemistry, the antibodies penetrate fixed cells or tissues to locate mCherry-tagged proteins within cellular compartments, providing spatial information about protein localization . Importantly, these antibodies don't cross-react with other fluorescent proteins like GFP, ensuring signal specificity when working with multiple fluorescent tags .

What applications are most suitable for mCherry monoclonal antibodies?

mCherry monoclonal antibodies have demonstrated efficacy across multiple experimental applications with varying sample types and detection methodologies:

These antibodies excel in detecting transgene expression in model organisms, verifying protein expression in transfected cells, and confirming successful protein tagging in recombinant protein studies . The high specificity allows researchers to confirm the presence of mCherry fusion proteins even in complex biological samples where the innate fluorescence of mCherry might be insufficient for direct visualization .

What are the critical factors for successful immunostaining with mCherry antibodies?

Successful immunostaining with mCherry antibodies depends on several critical factors:

Fixation protocol: The choice between paraformaldehyde (PFA), formalin, or other fixatives significantly impacts epitope preservation. For optimal results with mCherry antibodies, 4% paraformaldehyde fixation for 20 minutes at room temperature has proven effective for cultured cells . For tissue sections, 10% buffered formalin provides good antigen preservation while maintaining tissue architecture .

Blocking conditions: Effective blocking is essential to prevent non-specific binding. Protocols using either 5000 μg/ml bovine serum albumin (BSA) for 30 minutes at 22°C or 2% normal donkey serum (NDS) in 0.3% PBT have demonstrated optimal results in reducing background staining . The blocking agent should be compatible with both the tissue type and the host species of the primary and secondary antibodies.

Antibody dilution: Appropriate dilution is critical for balancing signal strength and specificity. For immunofluorescence applications, dilutions ranging from 1:6000-12000 have been validated, while immunohistochemistry may require higher concentrations (1:2000) . Each new experimental system requires optimization through dilution series testing.

Incubation conditions: Temperature and duration influence antibody penetration and binding kinetics. Successful protocols include incubation with primary antibody for 1 hour at 22°C or overnight at 4°C depending on sample thickness and antibody concentration .

Detection system: Selection of appropriate secondary antibodies or detection systems significantly impacts signal quality. For fluorescence applications, spectrally compatible secondary antibodies prevent bleed-through, while for chromogenic detection, enzyme-linked systems like HRP with substrates such as DAB provide stable signals .

How should western blot protocols be optimized for detecting mCherry fusion proteins?

Optimizing western blot protocols for mCherry fusion proteins requires attention to several key parameters:

Sample preparation: Effective lysis buffers typically contain detergents like NP-40 or Triton X-100 that solubilize membrane-associated proteins without denaturing the antibody epitopes. Complete protease inhibitor cocktails should be included to prevent degradation of fusion proteins .

Gel percentage selection: Since mCherry adds approximately 27-30 kDa to the fusion partner, selecting an appropriate gel percentage is essential. For smaller fusion partners (<50 kDa), 12% polyacrylamide gels offer optimal resolution, while larger fusion proteins benefit from 8-10% gels .

Transfer conditions: Semi-dry transfer at 15V for 30 minutes works well for proteins under 100 kDa, while larger fusion proteins benefit from wet transfer systems (30V overnight at 4°C) to ensure complete transfer .

Blocking and antibody conditions: Blocking with 5% non-fat dry milk in TBST for 1 hour at room temperature followed by overnight primary antibody incubation (1:1000-2000 dilution) at 4°C provides optimal results. Secondary antibody incubation should be limited to 1 hour at room temperature to minimize background .

Detection considerations: mCherry fusion proteins typically appear as distinct bands at molecular weights 27-30 kDa higher than the target protein alone. Multiple bands may indicate proteolytic cleavage, alternative splicing, or post-translational modifications of the fusion protein .

What controls are essential when working with mCherry antibodies?

Implementing appropriate controls is critical for experimental rigor when working with mCherry antibodies:

Positive controls:

• Recombinant mCherry protein serves as a definitive positive control, appearing as a distinct band at approximately 27-30 kDa in western blots

• Cells or tissues transfected with mCherry-expressing constructs provide system-specific positive controls that account for expression level variations and post-translational modifications

Negative controls:

• Wild-type or untransfected cells/tissues establish baseline signals and identify any non-specific binding

• GFP-expressing samples confirm antibody specificity against mCherry, as validated antibodies should not cross-react with GFP despite structural similarities between fluorescent proteins

Secondary antibody controls:

• Samples processed with secondary antibody alone (omitting primary antibody) identify non-specific binding from the secondary antibody system

• Isotype controls using non-specific antibodies of the same isotype (IgG1 for many mCherry monoclonal antibodies) help distinguish specific from non-specific binding

Loading controls:

• Simultaneous probing for housekeeping proteins (e.g., HSP60, β-actin) confirms equal loading and successful protein transfer in western blotting applications

• For imaging applications, nuclear counterstains like DAPI provide reference points for evaluating transfection efficiency and cellular morphology

How can researchers address weak or non-specific signals when using mCherry antibodies?

When encountering weak or non-specific signals with mCherry antibodies, systematic troubleshooting can identify and resolve the underlying issues:

For weak signals:

• Increase antibody concentration incrementally (e.g., from 1:2000 to 1:1000 for western blotting)

• Extend primary antibody incubation time (from 1 hour to overnight at 4°C) to enhance binding kinetics

• Modify antigen retrieval methods for fixed tissues (try citrate buffer pH 6.0 or EDTA buffer pH 9.0 with heat-induced epitope retrieval)

• Ensure target expression levels are sufficient by verifying transfection efficiency through direct mCherry fluorescence before immunostaining

• Switch detection systems to more sensitive options (e.g., TSA amplification for immunofluorescence or enhanced chemiluminescence for western blotting)

For non-specific signals:

• Increase blocking stringency using a combination of BSA (3-5%) and serum (5-10%) from the secondary antibody host species

• Add 0.1-0.3% Triton X-100 to blocking and antibody diluent solutions to reduce hydrophobic interactions

• Perform additional washing steps with higher salt concentration (up to 500 mM NaCl) to disrupt low-affinity binding

• Pre-absorb primary antibodies with wild-type lysates to remove antibodies that bind to endogenous proteins

• Validate antibody specificity using multiple applications (e.g., if non-specific bands appear in western blotting, compare with immunofluorescence patterns)

For high background:

• Reduce antibody concentration and ensure proper blocking before applying primary antibody

• Include 0.05-0.1% Tween-20 in wash buffers to remove weakly bound antibodies

• For tissues with high endogenous peroxidase activity, include a hydrogen peroxide quenching step (3% H₂O₂ for 10 minutes) before blocking

• Consider using fluorescently-labeled secondary antibodies instead of enzymatic detection to avoid signal amplification of non-specific binding

What are the potential cross-reactivity issues with mCherry antibodies?

Understanding potential cross-reactivity issues is essential for accurate interpretation of results when using mCherry antibodies:

Cross-reactivity with related fluorescent proteins:
The mFruits family includes several related red fluorescent proteins (mTomato, mStrawberry, mPlum) that share structural similarities with mCherry. While high-quality monoclonal antibodies like clone 1C51 demonstrate specificity for mCherry without recognizing GFP, cross-reactivity with other red fluorescent proteins should be evaluated when multiple fluorescent tags are used in the same experimental system .

Endogenous protein interference:
Some tissues exhibit autofluorescence in the red spectrum due to endogenous compounds (lipofuscin, porphyrins, flavins), which can confound immunofluorescence results. Control experiments with wild-type tissues processed identically are essential for distinguishing specific antibody binding from tissue autofluorescence .

Species-specific considerations:
While mCherry is exogenous to mammalian systems, antibody epitopes may occasionally share homology with endogenous proteins. For example, certain anti-mCherry antibodies have shown unexpected binding to stress granules or mitochondrial proteins in specific cell types. This potential for cross-reactivity underscores the importance of thorough validation with appropriate controls .

Technical cross-reactivity factors:
Secondary antibody cross-reactivity can occur when multiple primary antibodies from the same host species are used simultaneously. Using directly conjugated primary antibodies or sequential staining protocols with proper blocking steps between applications can minimize this technical issue .

How do fixation methods affect mCherry antibody detection in microscopy applications?

Fixation methodology significantly impacts both the native fluorescence of mCherry and the epitope accessibility for antibody binding:

Paraformaldehyde (PFA) fixation:

• 4% PFA for 20 minutes at room temperature preserves mCherry fluorescence while allowing antibody penetration

• Extended fixation (>24 hours) can reduce epitope accessibility, requiring more rigorous antigen retrieval

• PFA fixation maintains cellular architecture while providing good antibody accessibility in most applications

Methanol fixation:

• Methanol fixation (100% methanol, -20°C, 10 minutes) may diminish native mCherry fluorescence but can enhance antibody penetration for certain applications

• This method is particularly useful when membrane permeabilization is challenging with cross-linking fixatives

• The loss of native fluorescence means researchers must rely entirely on antibody detection rather than dual visualization methods

Formalin fixation and paraffin embedding (FFPE):

• 10% neutral buffered formalin followed by paraffin embedding requires heat-induced epitope retrieval for optimal antibody binding

• Antigen retrieval in citrate buffer (pH 6.0) for 20 minutes at 95-100°C significantly improves detection in FFPE tissues

• This method is compatible with chromogenic detection using peroxidase-conjugated secondary antibodies and DAB substrate

Glutaraldehyde inclusion:

• Even low concentrations of glutaraldehyde (0.05-0.1%) in fixatives can dramatically reduce antibody binding while preserving native fluorescence

• If electron microscopy correlation is planned, progressive lowering of temperature (PLT) methods with minimal glutaraldehyde can balance ultrastructural preservation with immunodetection

How can mCherry antibodies be used for multi-protein localization studies?

Multi-protein localization studies benefit from mCherry antibodies through several strategic approaches:

Sequential immunostaining protocols:
For co-localization of multiple proteins, sequential staining protocols overcome host species limitations. After completing the first round of primary and secondary antibody staining, a blocking step with excess unconjugated secondary antibody prevents cross-reactivity during subsequent rounds. This approach allows visualization of mCherry-tagged proteins alongside other cellular markers regardless of the primary antibody host species .

Multi-spectral imaging strategies:
When designing multi-protein visualization experiments, strategic selection of fluorophores with minimal spectral overlap is critical. mCherry antibodies can be detected with far-red secondary antibodies (e.g., Alexa Fluor 647), preserving shorter wavelengths for other targets and endogenous mCherry fluorescence. This approach enables triple or quadruple labeling when combined with conventional blue and green fluorophores .

Combining direct and indirect detection:
A hybrid approach leverages both the direct fluorescence of mCherry and antibody-based amplification. In samples with variable expression levels, direct mCherry fluorescence identifies high-expressing cells/regions, while antibody detection reveals low-expressing populations. This strategy is particularly valuable for lineage tracing experiments where expression levels may vary with cellular differentiation .

Correlative light and electron microscopy (CLEM):
For ultrastructural localization, mCherry antibodies can be detected with gold-conjugated secondary antibodies for electron microscopy. By first imaging the sample with fluorescence microscopy to identify regions of interest based on mCherry signal, then processing the same sample for immunogold labeling, researchers can precisely localize tagged proteins at the ultrastructural level .

What approaches enable quantitative analysis of protein expression using mCherry antibodies?

Quantitative analysis of protein expression using mCherry antibodies can be achieved through several methodologies:

Western blot densitometry:
Quantification of band intensity in western blots allows comparative analysis of mCherry fusion protein expression across different samples. For accurate quantification, standard curves using recombinant mCherry protein at known concentrations (5-100 ng) provide reference points for calculating absolute expression levels . Normalization to housekeeping proteins compensates for loading variations, enabling reliable relative quantification between experimental conditions .

Flow cytometry quantification:
Flow cytometric analysis offers single-cell resolution for quantifying mCherry-tagged protein expression across populations. Using mCherry antibodies with allophycocyanin-conjugated secondary antibodies provides sensitive detection independent of native mCherry fluorescence . This approach is particularly valuable for heterogeneous populations where expression varies between cells. Standard calibration beads with defined antibody binding capacity enable conversion of fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF) for absolute quantification .

Quantitative immunofluorescence microscopy:
Image-based quantification combines spatial information with expression level analysis. For accurate measurements, acquisition parameters must be standardized across all samples, with exposure times set to avoid saturation even in the highest-expressing samples . Background subtraction using control samples (wild-type or secondary-only) improves signal specificity, while inclusion of calibration standards in each imaging session compensates for day-to-day variations in microscope performance .

ELISA-based quantification:
For secreted mCherry fusion proteins, sandwich ELISA using anti-mCherry antibodies provides sensitive quantification in culture supernatants or biological fluids. Standard curves using purified mCherry protein enable absolute quantification down to 10-50 pg/ml depending on the detection system . This approach is particularly valuable for tracking secretion kinetics or comparing secretion efficiency across different experimental conditions .

What considerations are important when using mCherry antibodies in live animal models?

Working with mCherry antibodies in live animal models presents unique challenges and considerations:

Tissue penetration limitations:
Antibody penetration in intact tissues is limited by molecular size (approximately 150 kDa for IgG). For whole-mount applications, extended incubation times (24-72 hours) and tissue clearing methods improve detection depth . Alternatively, tissue sectioning at optimal thickness (5-20 μm depending on tissue density) balances structural integrity with antibody accessibility .

Perfusion fixation considerations:
For optimal preservation of mCherry-tagged proteins in animal tissues, transcardial perfusion with 4% paraformaldehyde yields superior results compared to immersion fixation . The perfusion rate and buffer composition should be optimized for each animal model, with PBS perfusion preceding fixative to remove blood and reduce background .

Background reduction strategies:
Endogenous immunoglobulins in animal tissues can interact with anti-mouse secondary antibodies, creating false-positive signals. This can be mitigated by using secondary antibodies specifically designed to recognize only the non-native primary antibody species (e.g., anti-mouse IgG that doesn't recognize endogenous mouse IgG) . Including blocking steps with unconjugated Fab fragments further reduces non-specific binding .

Comparative visualization approaches:
When working with transgenic animals expressing mCherry, comparing direct fluorescence with antibody-based detection provides complementary data. Direct fluorescence reveals the real-time distribution of the fusion protein, while antibody staining can detect accumulated protein in fixed tissues, even after partial degradation . This comparative approach is particularly valuable for studying protein turnover or trafficking in vivo .

How are mCherry antibodies being integrated with super-resolution microscopy techniques?

The integration of mCherry antibodies with super-resolution microscopy represents an expanding frontier in molecular imaging:

Single-molecule localization microscopy (SMLM):
For SMLM techniques like STORM or PALM, direct immunolabeling with fluorophore-conjugated mCherry antibodies provides superior localization precision compared to secondary detection methods. The smaller size of directly labeled primary antibodies (approximately 10-15 nm displacement from target) minimizes the "linkage error" that occurs with primary-secondary systems (20-30 nm displacement) . This approach enables co-localization studies with nanometer precision, particularly valuable for investigating protein interactions within subcellular compartments .

Stimulated emission depletion (STED) microscopy:
STED microscopy with mCherry antibodies typically employs secondary antibodies conjugated to photostable dyes optimized for depletion efficiency (e.g., STAR RED, ATTO 647N). Sample preparation requires careful optimization of fixation and permeabilization to maintain structural integrity at the nanoscale while ensuring antibody access to targets . This combination allows visualization of mCherry-tagged proteins with resolution approaching 30-50 nm .

Expansion microscopy compatibility:
Physical expansion of specimens through polymer embedding and swelling provides another super-resolution approach compatible with standard fluorescence microscopes. mCherry antibodies have demonstrated compatibility with expansion microscopy protocols, maintaining epitope recognition after the chemical treatments required for polymer linkage and protein digestion . This technique enables effective resolution enhancement of 4-10 fold while preserving the multiplexing capabilities of antibody-based detection .

Correlative light and electron microscopy (CLEM):
Advanced CLEM approaches combine super-resolution fluorescence imaging with electron microscopy of the same sample. For these applications, mCherry antibodies can be detected with both fluorescent and electron-dense markers (e.g., fluoronanogold secondary antibodies) . This dual-detection strategy enables precise registration between super-resolution fluorescence images and ultrastructural data .

What are the emerging applications for mCherry antibodies in extracellular vesicle research?

Extracellular vesicle (EV) research represents an emerging application area for mCherry antibodies:

EV cargo tracking and verification:
mCherry-tagged proteins incorporated into EVs can be detected and quantified using anti-mCherry antibodies in immunoblotting or ELISA assays. This approach enables verification of specific protein loading into EVs and quantitative assessment of cargo incorporation efficiency . The high sensitivity of antibody detection overcomes limitations associated with direct fluorescence visualization of EVs, which typically contain few copies of any given protein .

Immunoaffinity isolation of specific EV subpopulations:
When EV surface proteins are tagged with mCherry, anti-mCherry antibodies can be immobilized on magnetic beads or affinity matrices to isolate specific EV subpopulations. This approach enables selective enrichment of EVs containing particular membrane proteins, facilitating downstream analysis of associated cargo molecules . The specificity of monoclonal antibodies minimizes contamination with non-target vesicles .

EV uptake and trafficking studies:
Antibody-based detection of mCherry-tagged proteins in recipient cells after EV uptake provides sensitive tracking of EV fate. By combining direct fluorescence observation of mCherry (indicating intact protein) with antibody detection (which can recognize even partially degraded protein), researchers can monitor both EV internalization and subsequent cargo processing . This dual-detection approach enables temporal resolution of cargo delivery and degradation kinetics .

Multiplexed EV characterization:
Flow cytometric analysis of EVs using combinations of antibodies against mCherry (for tagged cargo) and endogenous EV markers enables high-throughput characterization of heterogeneous EV populations. This approach allows correlation between cargo content and vesicle origin, particularly valuable for understanding selective cargo sorting into different EV subtypes .