mCherry Mouse Polyclonal Antibody

What is mCherry and why is it used as a fluorescent marker in molecular biology?

mCherry is a monomeric fluorescent protein derived from proteins originally isolated from Cnidarians (jellyfish, sea anemones, and corals). It was developed through multiple cycles of mutation, directed modification, and evolutionary selection from the Discosoma red (DsRed) protein . As a fluorescent marker, mCherry offers several advantages for molecular biology applications:

mCherry exhibits peak absorption at 587 nm and emission at 610 nm, allowing for detection in the red spectrum . Its monomeric nature makes it an excellent choice for fusion protein studies, as it minimizes aggregation issues commonly encountered with other fluorescent proteins . Additionally, mCherry demonstrates superior photostability compared to other monomeric fluorophores, making it particularly valuable for long-duration imaging experiments .

The protein serves as a tracer in transfection and transgenic experiments, allowing researchers to visualize protein localization, expression, and dynamics in living cells and organisms .

How does the mCherry Mouse Polyclonal Antibody specifically detect mCherry-tagged proteins?

The mCherry Mouse Polyclonal Antibody is specifically raised against recombinant mCherry protein . These polyclonal antibodies recognize multiple epitopes on the mCherry protein structure, providing robust detection capabilities. When proteins are tagged with mCherry, the antibody binds to the mCherry portion of the fusion protein, enabling visualization and analysis of the tagged protein of interest .

The specificity of these antibodies is typically validated through experiments with transfected and non-transfected cells. For example, immunofluorescence studies show that the antibody staining is only observed in cells expressing mCherry, with no cross-reactivity in non-transfected cells . Western blot analyses similarly demonstrate specific binding to mCherry-tagged proteins at the expected molecular weight, with no bands detected in control samples lacking mCherry expression .

What are the validated applications for mCherry Mouse Polyclonal Antibody?

Based on the search results, mCherry Mouse Polyclonal Antibodies have been validated for multiple applications in molecular and cellular biology research:

These antibodies have demonstrated high specificity across these applications, with validated protocols showing minimal background and strong signal-to-noise ratios when used at the recommended dilutions .

What controls should be included when using mCherry Mouse Polyclonal Antibody in immunofluorescence experiments?

When performing immunofluorescence with mCherry Mouse Polyclonal Antibody, several essential controls should be included to ensure reliable and interpretable results:

• Positive Control: Cells transfected with an mCherry expression vector serve as the ideal positive control. These cells should show specific staining with both the endogenous mCherry fluorescence (red) and the antibody staining (typically visualized with a different color fluorophore such as green) .

• Negative Control: Non-transfected cells processed identically to experimental samples should show no specific staining with the mCherry antibody. This control helps establish the specificity of the antibody and identifies any potential non-specific binding .

• Secondary Antibody-Only Control: Samples treated with secondary antibody but without the primary mCherry antibody help identify background fluorescence caused by non-specific binding of the secondary antibody.

• Blocking Peptide Control: Pre-incubation of the antibody with purified mCherry protein before staining can confirm specificity, as this should abolish specific staining.

• Co-localization Control: When assessing whether the antibody truly recognizes mCherry, analyzing the overlap between the antibody signal and the direct fluorescence from mCherry is crucial. In properly validated experiments, these signals should show strong co-localization .

How should I optimize Western blot protocols for detecting mCherry-tagged proteins?

Optimizing Western blot protocols for mCherry-tagged proteins requires attention to several key factors:

• Sample Preparation: For cell extracts, lysis buffers containing phosphate buffered saline with 150mM NaCl and protease inhibitors are recommended . Solubilize adherent cells at a concentration of approximately 2×10^7 cells/mL in cell extraction buffer .

• Protein Loading: Load 10-20 μg of total protein per lane. For quantitative analyses, consider preparing a standard curve using purified mCherry protein.

• Antibody Dilution: The recommended working dilution for Western blot is 1:5,000, though this may need adjustment based on specific experimental conditions and the expression level of your target protein .

• Blocking Conditions: Use 5% non-fat dry milk or bovine serum albumin in TBST (Tris-buffered saline with 0.1% Tween-20) for blocking to minimize background signal.

• Incubation Times: For primary antibody, incubate overnight at 4°C or for 2 hours at room temperature. For secondary antibody, a 1-hour incubation at room temperature is typically sufficient.

• Detection Method: Enhanced chemiluminescence (ECL) offers good sensitivity for most applications. For low-abundance proteins, consider using more sensitive detection methods or signal amplification strategies.

• Expected Band Size: Remember that the mCherry tag adds approximately 27 kDa to your protein of interest . Factor this into your size estimation when analyzing results.

What are the key considerations for using mCherry Mouse Polyclonal Antibody in immunoprecipitation experiments?

When conducting immunoprecipitation (IP) experiments with mCherry Mouse Polyclonal Antibody, consider these critical factors:

• Antibody Amount: Use approximately 1-5 μg of antibody per 100-500 μg of total protein, with a recommended dilution of 1:100 for IP applications .

• Pre-clearing Step: To reduce non-specific binding, pre-clear lysates with protein G beads before adding the mCherry antibody.

• Lysis Buffer Composition: Use a gentle lysis buffer that preserves protein-protein interactions while effectively solubilizing membrane proteins. Common components include 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), and protease/phosphatase inhibitors.

• Incubation Conditions: For optimal antibody-antigen binding, incubate the antibody with the lysate overnight at 4°C with gentle rotation.

• Washing Stringency: The stringency of washes impacts the signal-to-noise ratio. More stringent washes (higher salt, addition of detergents) reduce background but may also reduce specific signal. Optimize the number and composition of washes based on your specific experiment.

• Elution Method: For most applications, boiling in SDS sample buffer effectively elutes bound proteins. For applications requiring native protein, consider elution with excess mCherry peptide.

• Controls: Always include a negative control using non-specific IgG from the same species as the mCherry antibody to identify non-specific pull-down products.

How can I use mCherry Mouse Polyclonal Antibody in multi-color immunofluorescence experiments?

Multi-color immunofluorescence with mCherry Mouse Polyclonal Antibody requires careful planning to avoid spectral overlap and maximize signal separation:

• Fluorophore Selection: When designing multi-color experiments, consider that mCherry itself emits red fluorescence (peak emission at 610 nm) . Choose secondary antibodies conjugated to fluorophores in different spectral regions. Common combinations include:

  • Anti-mouse secondary with Alexa Fluor 488 (green) or Alexa Fluor 647 (far red) to detect the mCherry antibody

  • Other primary antibodies from different host species (rabbit, goat, etc.) for co-staining

• Sequential Staining Protocol: For complex multi-color experiments, consider a sequential staining approach:

  • First round: mCherry antibody + fluorescent secondary antibody

  • Blocking step with excess mouse IgG

  • Second round: Additional primary antibodies + species-specific secondary antibodies

• Image Acquisition Settings: Acquire images sequentially using channel-specific settings to minimize bleed-through. Include single-stained controls to establish proper exposure settings and confirm absence of cross-talk between channels.

• Analysis of Co-localization: When analyzing co-localization between mCherry and other markers, it's important to note that the antibody signal (typically visualized in green) should overlap with the direct mCherry fluorescence (red), producing a yellow signal in merged images .

• Spectral Unmixing: For confocal microscopy with multiple fluorophores, spectral unmixing algorithms can separate overlapping emission spectra and improve signal specificity.

What strategies can improve detection of low-abundance mCherry-tagged proteins in complex samples?

Detecting low-abundance mCherry-tagged proteins presents challenges that can be addressed through several optimization strategies:

• Signal Amplification Systems: Consider tyramide signal amplification (TSA) or other enzymatic amplification methods to boost signal intensity while maintaining specificity.

• Sample Enrichment: For cell extracts, concentrate proteins of interest through subcellular fractionation or affinity purification before analysis. The mCherry SimpleStep ELISA can be used to quantify concentration of mCherry in samples prior to other analyses .

• Optimized Sample Preparation:

  • For tissue sections: Optimal antigen retrieval methods (heat-induced or enzymatic) can significantly improve epitope accessibility

  • For Western blot: Use gradient gels and extended transfer times for high molecular weight fusion proteins

• Extended Antibody Incubation: Increase primary antibody concentration (up to 1:100 dilution) and extend incubation time (overnight at 4°C) to maximize binding to low-abundance targets .

• Sensitive Detection Methods:

  • For Western blot: Use high-sensitivity chemiluminescent substrates or fluorescent secondary antibodies with digital imaging

  • For microscopy: Consider using photomultiplier tubes (PMTs) with higher gain settings or electron-multiplying CCD cameras

• Background Reduction: Implement more stringent blocking protocols using a combination of BSA, normal serum, and mild detergents to improve signal-to-noise ratio. For immunofluorescence, include an autofluorescence quenching step.

What are common issues encountered with mCherry Mouse Polyclonal Antibody and how can they be resolved?

Researchers working with mCherry Mouse Polyclonal Antibody may encounter several common challenges. Here are solutions for typical problems:

• Weak or No Signal in Western Blot:

  • Increase antibody concentration (try 1:1,000 instead of 1:5,000)

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

  • Verify protein transfer efficiency with reversible protein stain

  • Check expression levels of mCherry-tagged protein

  • Ensure sample buffer doesn't contain excessive reducing agents that might destroy epitopes

• High Background in Immunofluorescence:

  • Optimize blocking (try 5% BSA with 0.3% Triton X-100)

  • Increase washing steps (use at least 3-5 washes of 5 minutes each)

  • Dilute primary antibody further (try 1:500 instead of 1:200)

  • Include 0.1% Tween-20 in antibody dilution buffer

  • Pre-adsorb antibody against fixed, untransfected cells

• Cross-Reactivity Issues:

  • Use pre-adsorbed antibodies that have been treated to remove reactivity against human, mouse, or rat serum proteins

  • Include additional blocking steps with normal serum from the same species as your sample

  • Validate antibody specificity using knockout or non-expressing controls

• Inconsistent Results Between Experiments:

  • Standardize fixation protocols (fixation method and duration significantly impacts epitope availability)

  • Use the same lot of antibody when possible

  • Implement positive controls in each experiment

  • Create detailed protocols with standardized reagent preparations

• Poor Signal in Immunoprecipitation:

  • Increase antibody amount (up to 5 μg per reaction)

  • Extend binding time (overnight at 4°C)

  • Use gentler lysis conditions to preserve epitopes

  • Cross-link antibody to beads to prevent antibody co-elution

How can I validate the specificity of my mCherry Mouse Polyclonal Antibody prior to critical experiments?

Thorough validation of antibody specificity is essential before conducting significant experiments. Here's a systematic approach:

• Western Blot Validation:

  • Run paired samples of cells expressing and not expressing mCherry-tagged proteins

  • Verify a single band at the expected molecular weight (mCherry adds ~27 kDa)

  • Perform peptide competition assay (pre-incubate antibody with purified mCherry)

• Immunofluorescence Validation:

  • Compare antibody staining pattern with direct mCherry fluorescence in transfected cells

  • Confirm co-localization between antibody signal and direct mCherry signal

  • Verify absence of staining in non-transfected cells

• Flow Cytometry Validation:

  • Analyze mixed populations of mCherry-expressing and non-expressing cells

  • Confirm correlation between direct mCherry fluorescence and antibody staining

• Knockout/Knockdown Controls:

  • If possible, test the antibody on samples where mCherry expression has been eliminated

  • This provides the most stringent specificity control

• Batch Testing Protocol:

  • When receiving a new antibody lot, perform a standard validation protocol

  • Document assay conditions and results for reference

• Dilution Series Testing:

  • Test a range of antibody dilutions to identify optimal signal-to-noise ratio

  • Create a standardized dilution curve for reference

What factors affect epitope recognition by mCherry Mouse Polyclonal Antibody in fixed samples?

Several factors can impact epitope recognition in fixed samples, which is critical for immunofluorescence and immunohistochemistry applications:

• Fixation Method Impact:

  • Paraformaldehyde (4%): Preserves mCherry epitopes well while maintaining cellular architecture

  • Methanol: May disrupt some conformational epitopes but can improve access to others

  • Glutaraldehyde: Provides excellent structural preservation but can reduce antibody accessibility and increase autofluorescence

  • Fresh frozen samples: Offer good epitope preservation but poorer morphology

• Fixation Duration:

  • Over-fixation can mask epitopes through excessive cross-linking

  • Under-fixation may result in poor morphology and sample loss

  • Recommended fixation: 15-20 minutes with 4% paraformaldehyde at room temperature

• Antigen Retrieval Methods:

  • Heat-induced epitope retrieval (HIER): Can recover epitopes masked by fixation

  • Enzymatic retrieval: Gentle protease treatment may improve accessibility

  • pH considerations: Test both acidic (citrate buffer, pH 6.0) and basic (Tris-EDTA, pH 9.0) retrieval solutions

• Permeabilization Factors:

  • Membrane permeabilization is essential for antibody access to intracellular epitopes

  • Common agents include Triton X-100 (0.1-0.3%), Tween-20 (0.1%), or saponin (0.1%)

  • Permeabilization duration affects antibody penetration and background

• Blocking Protocol Influence:

  • Insufficient blocking leads to high background

  • Excessive blocking may mask epitopes

  • Optimal blocking: 5-10% normal serum from the secondary antibody species plus 1-3% BSA

How can mCherry Mouse Polyclonal Antibody be used for quantitative protein expression analysis?

Quantitative analysis of protein expression using mCherry Mouse Polyclonal Antibody requires methodological rigor across several techniques:

• Quantitative Western Blot:

  • Create a standard curve using purified mCherry protein at known concentrations

  • Use digital imaging systems rather than film for wider dynamic range

  • Include loading controls (β-actin, GAPDH) for normalization

  • For accurate quantification, operate within the linear range of detection

  • Analysis software: Use ImageJ or specialized Western blot quantification software with background subtraction

• ELISA-Based Quantification:

  • The mCherry SimpleStep ELISA Kit provides a sensitive method for quantitative measurement

  • Prepare standards using the provided mCherry protein (9 standards from 0-4,000 pg/mL)

  • The assay demonstrates high sensitivity with a minimum detectable dose of 6.9 pg/mL

  • Sample types compatible with this method include cell culture supernatants and cell extracts

• Flow Cytometry Quantification:

  • Use calibration beads with known quantities of fluorophore to establish a standard curve

  • Analyze mean fluorescence intensity (MFI) to determine relative expression levels

  • Consider compensation for spectral overlap when using multiple fluorophores

• Immunofluorescence Quantification:

  • Use consistent image acquisition settings across all samples

  • Apply appropriate background subtraction methods

  • Measure integrated density or mean pixel intensity within defined regions of interest

  • Include reference standards in each imaging session

• Assay Validation Parameters:

  • Linearity: Verify linear relationship between protein concentration and signal intensity

  • Precision: Assess intra-assay and inter-assay variation (coefficient of variation <15%)

  • Recovery: Spike samples with known amounts of purified mCherry to assess recovery percentage

What are the considerations for using mCherry Mouse Polyclonal Antibody in tissue clearing and 3D imaging applications?

Using mCherry Mouse Polyclonal Antibody in tissue clearing and 3D imaging requires specific adaptations:

• Compatibility with Clearing Methods:

  • CLARITY-based methods: mCherry antibodies maintain reactivity; use 1:100 dilution with extended incubation (3-7 days)

  • Organic solvent methods (e.g., iDISCO): Test antibody compatibility as organic solvents may denature some antibodies

  • Aqueous-based methods (Scale, SeeDB): Generally compatible with most antibodies but may require increased concentration

• Penetration Optimization:

  • Use higher antibody concentrations (1:50-1:100) than for thin sections

  • Extend incubation times (days rather than hours)

  • Consider using Fab fragments for better penetration in dense tissues

  • Include 0.1-0.5% Triton X-100 and carrier proteins to improve penetration

• 3D Imaging Considerations:

  • Signal-to-background ratio becomes more critical in 3D imaging

  • Implement more rigorous blocking protocols to reduce non-specific binding

  • Include multiple washing steps (3-5 days) with gentle agitation

  • Consider using light sheet microscopy for faster acquisition with reduced photobleaching

• Multiplexing Strategies:

  • Sequential immunolabeling may be necessary for multiple antigens

  • Allow sufficient elution/washing between rounds of staining

  • Choose fluorophores with minimal spectral overlap

• Sample Preparation Modifications:

  • Perfusion fixation improves antibody penetration compared to immersion fixation

  • Optimize fixation time carefully - under-fixation leads to poor morphology while over-fixation reduces antibody penetration

  • Consider vibratome sectioning to create thicker sections (100-300 μm) prior to clearing for improved antibody access

How does the performance of Mouse Polyclonal anti-mCherry compare with antibodies from other host species?

Different host species antibodies against mCherry offer distinct advantages and limitations:

Performance comparison in specific applications:

• Western Blot Performance:

  • Mouse polyclonal: Effective at 1:5,000 dilution with good sensitivity

  • Rabbit polyclonal: Often usable at higher dilutions (1:5,000-1:10,000)

  • Chicken polyclonal: May require lower dilutions but offers excellent specificity

• Immunofluorescence Sensitivity:

  • Mouse polyclonal: Works well at 1:200 dilution in non-mouse tissues

  • Rabbit polyclonal: Often preferred for mouse tissue sections to avoid background

  • Chicken polyclonal: Excellent for multi-color staining with minimal cross-reactivity

• Immunoprecipitation Efficiency:

  • Mouse and rabbit polyclonals typically demonstrate comparable IP efficiency

  • Choice often depends on compatibility with other antibodies in downstream applications

How can mCherry Mouse Polyclonal Antibody be integrated into advanced imaging techniques like super-resolution microscopy?

Incorporating mCherry Mouse Polyclonal Antibody into super-resolution microscopy requires specific considerations:

• STED Microscopy Integration:

  • Use secondary antibodies conjugated to STED-compatible dyes (STAR635P, ATTO647N)

  • Optimize fixation to minimize sample shrinkage and epitope masking

  • Consider using smaller probes (Fab fragments) for improved resolution

  • Expect resolution improvements from ~200 nm (confocal) to ~30-80 nm

• STORM/PALM Applications:

  • Select secondary antibodies with appropriate photoswitchable fluorophores (Alexa Fluor 647)

  • Control labeling density to achieve optimal single-molecule localization

  • Use appropriate imaging buffers (oxygen scavenging systems with thiol additives)

  • Implement drift correction strategies for long acquisition times

• Expansion Microscopy Considerations:

  • Test antibody retention during the expansion process

  • May require post-expansion staining for optimal results

  • Use fluorophores stable in expansion gel conditions

  • Account for dilution of signal due to physical expansion

• Sample Preparation Modifications:

  • Thinner sections (~10 μm) may be preferable for some super-resolution techniques

  • More stringent fixation protocols to minimize structural deformation

  • Consider the use of post-fixation steps to stabilize antibody binding

• Validation Approaches:

  • Compare super-resolution images with conventional microscopy to confirm biological relevance

  • Include appropriate resolution standards to verify performance

  • Implement quantitative analysis of resolution improvement

What are the considerations for using mCherry antibodies in combination with proximity labeling techniques?

Integrating mCherry antibodies with proximity labeling offers powerful approaches for studying protein interactions and localization:

• BioID/TurboID Applications:

  • mCherry-tagged BioID/TurboID fusion proteins can be validated using anti-mCherry antibodies

  • Verify proper subcellular localization of fusion proteins by immunofluorescence

  • Confirm expression levels and fusion protein integrity by Western blot

  • Use in parallel with streptavidin detection to correlate enzyme localization with biotinylation patterns

• APEX2 Proximity Labeling:

  • mCherry-APEX2 fusions allow visualization of the peroxidase location

  • Antibody staining can confirm expression and localization prior to EM processing

  • Can be used to correlate fluorescence microscopy with electron microscopy data

  • Verify proper targeting and expression level before performing DAB reactions

• Split-BioID/APEX Applications:

  • mCherry tags on split constructs help visualize co-expression and co-localization

  • Antibody detection confirms proper expression of both construct components

  • Important for validating negative results (confirming both components are expressed)

• Technical Considerations:

  • Fixation must preserve both mCherry epitopes and biotin/DAB reaction products

  • Mild permeabilization conditions help retain biotinylated proteins

  • Sequential detection protocols may be necessary (detect mCherry first, then biotinylated proteins)

  • Pre-adsorbed antibodies minimize cross-reactivity in complex multi-labeling experiments