MRAS Antibody, FITC conjugated

What is MRAS and what cellular functions does it regulate?

MRAS (Muscle RAS Oncogene Homolog) is a member of the RAS superfamily of GTP-binding proteins. These proteins function as membrane-anchored intracellular signal transducers responsible for a variety of normal cellular functions. MRAS specifically participates in reorganization of the actin cytoskeleton and is involved in focal adhesion formation. Additionally, it can activate MAPK signaling pathways, making it an important molecule in cellular communication networks . The dysregulation of RAS family proteins, including MRAS, is oncogenically significant as they are activated in a substantial fraction of tumors, highlighting their importance in cancer research .

What are the key characteristics of FITC-conjugated antibodies?

FITC (Fluorescein Isothiocyanate) conjugated antibodies are primary antibodies directly labeled with the FITC fluorophore using established crosslinking protocols . These conjugated antibodies eliminate the need for secondary antibody incubation steps in immunofluorescence experiments, simplifying workflow and reducing background. FITC-conjugated antibodies are typically supplied in Phosphate-Buffered Saline (PBS) with 0.01% sodium azide as a preservative . The FITC fluorophore has an excitation maximum at approximately 495 nm and an emission maximum at around 520 nm, producing bright green fluorescence when excited with appropriate wavelengths of light.

What applications are supported by MRAS Antibody, FITC conjugated?

MRAS Antibody, FITC conjugated supports multiple research applications including:

• Immunofluorescence (IF) microscopy for both cell cultures and tissue sections

• Immunohistochemistry on paraffin-embedded (IHC-P) samples with recommended dilutions of 1:50-200

• Flow cytometry for analyzing MRAS expression in cell populations

• Fluorescence-based co-localization studies

• Live cell imaging (depending on cell permeability)

The polyclonal nature of commercially available MRAS antibodies makes them versatile for detecting the target protein across different experimental platforms .

What species reactivity is available for MRAS Antibody, FITC conjugated?

Commercial MRAS Antibody, FITC conjugated products demonstrate varied species reactivity. Some antibodies like ABIN7167212 are specific to human MRAS , while others (such as BS-1882R-FITC) offer broader reactivity against human, mouse, and rat MRAS proteins . When selecting an antibody for your research, it is crucial to verify the specific species reactivity to ensure compatibility with your experimental model. Cross-reactivity information is typically provided in the product datasheet and should be carefully reviewed before purchase.

What is the recommended storage protocol for MRAS Antibody, FITC conjugated?

For optimal preservation of FITC-conjugated antibodies, including MRAS Antibody:

• Store at -20°C in the dark to prevent photobleaching of the fluorophore

• Avoid repeated freeze-thaw cycles as this may denature the antibody

• Storage in frost-free freezers is not recommended due to temperature fluctuations

• For short-term storage (1-2 weeks), antibodies can be kept at 4°C protected from light

• Consider preparing small aliquots of the antibody solution to minimize freeze-thaw cycles

Proper storage conditions maintain antibody integrity and fluorescence intensity, ensuring consistent experimental results over time.

How can I validate the specificity of MRAS Antibody, FITC conjugated?

Validation of MRAS Antibody, FITC conjugated specificity is critical for generating reliable research data. A comprehensive validation approach includes:

• Positive and negative controls:

  • Use cell lines known to express (positive control) or not express (negative control) MRAS

  • Include MRAS-knockout cells as definitive negative controls

  • Compare staining patterns with published literature

• Peptide competition assay:

  • Pre-incubate the antibody with the immunogen (recombinant Human Ras-related protein M-Ras protein (1-205aa))

  • A specific antibody will show significantly reduced or eliminated staining

• Orthogonal method comparison:

  • Confirm MRAS detection with alternative methods like Western blotting or qPCR

  • Compare results from different antibody clones targeting different MRAS epitopes

• Isotype control experiments:

  • Include rabbit IgG-FITC at the same concentration to assess non-specific binding

  • This control helps distinguish specific signal from background fluorescence

• Subcellular localization analysis:

  • Verify that staining matches the expected membrane/cytoskeletal association pattern for MRAS

  • Co-localization with known interacting partners can further support specificity

What are the optimal fixation and permeabilization protocols for MRAS immunofluorescence studies?

The detection of membrane-associated proteins like MRAS requires careful consideration of fixation and permeabilization methods:

Permeabilization options:

• For PFA-fixed samples:

  • 0.1-0.2% Triton X-100 (5-10 minutes)

  • 0.05-0.1% Saponin (gentler alternative that maintains membrane structure)

  • Digitonin (0.01-0.05%) for selective plasma membrane permeabilization

• Critical considerations:

  • Over-permeabilization may disrupt MRAS membrane localization

  • Under-permeabilization may prevent antibody access to intracellular epitopes

  • Multiple gentle permeabilization steps may be more effective than a single harsh treatment

• Recommended optimization:

  • Test multiple fixation/permeabilization combinations

  • Evaluate signal intensity, background, and preservation of expected MRAS localization

How can I optimize signal-to-noise ratio when using MRAS Antibody, FITC conjugated?

Achieving optimal signal-to-noise ratio is essential for accurate interpretation of MRAS localization and expression levels:

• Antibody dilution optimization:

  • Perform a dilution series (starting with 1:50-1:200 as recommended)

  • Identify the concentration that provides maximum specific signal with minimal background

• Blocking optimization:

  • Use 5-10% serum from the same species as your secondary antibody

  • Consider specialized blocking solutions containing BSA, glycine, and Tween-20

  • For tissues with high autofluorescence, include 0.1-0.3% Sudan Black B in the blocking solution

• Washing optimization:

  • Increase washing duration (3-5 washes of 5-10 minutes each)

  • Use PBS with 0.05-0.1% Tween-20 to reduce non-specific binding

  • Consider adding 0.1% BSA to washing buffer to minimize background

• Autofluorescence reduction:

  • Treat samples with 0.1-1% sodium borohydride before blocking

  • Use TrueBlack® or similar reagents to quench naturally occurring tissue autofluorescence

  • Consider spectral unmixing during image acquisition to separate FITC signal from autofluorescence

• Counterstain selection:

  • Choose counterstains spectrally distinct from FITC (e.g., DAPI for nuclei, rhodamine-phalloidin for actin)

  • Ensure counterstain concentrations don't overwhelm the FITC signal

What approaches can be used to analyze MRAS interactions with MAPK signaling components?

Investigating MRAS interactions with MAPK signaling components requires sophisticated experimental approaches:

• Co-immunoprecipitation with FITC detection:

  • Use anti-MRAS antibodies to pull down protein complexes

  • Detect co-precipitated MAPK components using appropriate antibodies

  • The FITC-conjugated MRAS antibody can be used to verify successful immunoprecipitation of MRAS

• Proximity ligation assays (PLA):

  • Combine MRAS Antibody, FITC conjugated with antibodies against suspected interaction partners

  • Secondary antibodies with attached DNA strands allow amplification of signals when proteins are in close proximity

  • This provides spatial information about MRAS-MAPK component interactions

• FRET (Förster Resonance Energy Transfer) analysis:

  • Pair FITC-conjugated MRAS antibody (donor) with antibodies against interaction partners labeled with appropriate acceptor fluorophores

  • Measure energy transfer as evidence of molecular proximity

  • Requires careful controls to account for spectral overlap

• Live cell imaging approaches:

  • For cell-permeable antibody fragments or in permeabilized cells

  • Monitor dynamic changes in MRAS-MAPK interactions following stimulation

  • Combine with pharmacological inhibitors to dissect pathway dependencies

How can I analyze the quality and purity of MRAS Antibody, FITC conjugated preparations?

Quality control of MRAS Antibody, FITC conjugated is essential for experimental reproducibility:

• Spectrophotometric analysis:

  • Measure absorbance at 280 nm (protein) and 495 nm (FITC)

  • Calculate fluorophore-to-protein ratio to assess conjugation efficiency

  • Optimal F/P ratios typically range from 3:1 to 7:1 for most applications

• SDS-PAGE analysis:

  • Run reduced and non-reduced samples to assess antibody integrity

  • Look for appropriate band patterns (heavy and light chains)

  • Visualize fluorescence directly in-gel before protein staining

• Size exclusion chromatography:

  • Detect aggregation or fragmentation of the conjugated antibody

  • Compare with unconjugated antibody standards

• Mass spectrometry analysis:

  • Similar to approaches used for bispecific antibodies

  • Can provide precise determination of conjugation sites and degree of labeling

• Functional testing:

  • Flow cytometry with cells expressing different levels of MRAS

  • Compare staining intensity with benchmark lots of the same antibody

Most commercial MRAS Antibody, FITC conjugated products undergo purification by affinity chromatography, typically achieving >95% purity as determined by Protein G purification .

What controls should be included in experiments using MRAS Antibody, FITC conjugated?

A robust experimental design requires appropriate controls:

• Antibody controls:

  • Isotype control: Rabbit IgG-FITC at equivalent concentration

  • Unstained controls to assess autofluorescence

  • Secondary-only controls (if using indirect methods in parallel)

• Biological controls:

  • MRAS knockdown/knockout samples

  • Cell lines with known differential MRAS expression

  • Stimulated vs. unstimulated cells (for MAPK pathway activation studies)

• Technical controls:

  • Single-color controls for spectral compensation

  • Blocking peptide control (pre-adsorption with immunogen)

  • Dilution series to confirm signal specificity

• Processing controls:

  • Fixed vs. unfixed samples to assess fixation artifacts

  • Different permeabilization methods to optimize epitope access

  • Counterstain-only samples to assess bleed-through

How can I troubleshoot weak or absent MRAS staining?

If MRAS immunofluorescence signal is weak or absent, consider these troubleshooting approaches:

• Epitope accessibility issues:

  • Try alternative fixation methods (PFA, methanol, or acetone)

  • Increase permeabilization time or concentration

  • Consider antigen retrieval methods (heat-induced or enzymatic)

• Antibody-related issues:

  • Verify antibody concentration (try higher concentrations)

  • Check for antibody degradation (fluorescence loss)

  • Confirm species reactivity matches your sample

• Sample-related issues:

  • Verify MRAS expression in your sample type

  • Consider the activation state of MRAS (GTP-bound vs. GDP-bound)

  • Assess whether sample processing affected protein retention

• Detection system limitations:

  • Optimize microscope settings (exposure time, gain)

  • Use signal amplification methods if needed

  • Consider photobleaching during long imaging sessions

What quantification methods are appropriate for MRAS Antibody, FITC conjugated studies?

Quantitative analysis of MRAS expression and localization can be approached through:

• Immunofluorescence quantification:

  • Mean fluorescence intensity measurement in defined regions of interest

  • Colocalization coefficients with membrane or cytoskeletal markers

  • Segmentation-based quantification of MRAS-positive structures

• Flow cytometry analysis:

  • Mean/median fluorescence intensity of cell populations

  • Percentage of MRAS-positive cells

  • Correlation with other markers in multiparameter analysis

• Western blot correlation:

  • Validate immunofluorescence quantification with parallel western blot analysis

  • Compare relative expression levels between methods

• Image analysis software recommendations:

  • ImageJ/FIJI with appropriate plugins for colocalization and intensity analysis

  • CellProfiler for automated high-throughput analysis

  • Specialized commercial software for advanced analyses

How can I design co-localization experiments with MRAS Antibody, FITC conjugated?

Co-localization studies require careful planning to avoid artifacts and generate meaningful data:

How does FITC conjugation affect antibody performance compared to unconjugated alternatives?

FITC conjugation introduces both advantages and limitations that researchers should consider:

• Performance impacts:

  • Slight reduction in binding affinity may occur due to conjugation

  • Direct detection eliminates secondary antibody cross-reactivity concerns

  • Faster protocol with fewer washing steps

  • Potential for higher background in some tissues

• Technical considerations:

  • FITC is sensitive to photobleaching (more than newer fluorophores)

  • FITC fluorescence is pH-sensitive (optimal at pH 8.0-9.0)

  • Cannot be combined with HRP-based amplification methods

  • Limited options for signal amplification compared to unconjugated antibodies

• Alternative approaches when needed:

  • Consider two-step detection with unconjugated primary for maximum sensitivity

  • Alternative fluorophores (Alexa Fluor 488) offer greater photostability

  • Tyramide signal amplification may be necessary for low abundance targets

What factors can lead to misinterpretation of MRAS immunofluorescence data?

Awareness of potential artifacts and limitations is crucial for accurate data interpretation:

• Technical artifacts:

  • Autofluorescence from fixatives, especially glutaraldehyde

  • Edge effects in tissue sections

  • Nuclear trapping of antibodies (non-specific)

  • Mounting medium incompatibility causing signal quenching

• Biological considerations:

  • MRAS activation state may affect epitope accessibility

  • Post-translational modifications might mask antibody binding sites

  • Expression levels may vary significantly between cell types

  • Localization changes during cell cycle or activation state

• Analytical pitfalls:

  • Over-saturation of digital images leading to false co-localization

  • Threshold selection biases in quantification

  • Inadequate sampling of heterogeneous tissues

  • Misattribution of punctate background as specific signal

• Documentation recommendations:

  • Include both overlay and single-channel images in publications

  • Provide details on imaging parameters and processing steps

  • Show representative images of controls

  • Use consistent scaling for comparative analyses

What are the advantages and limitations of polyclonal vs. monoclonal MRAS antibodies for FITC conjugation?

The choice between polyclonal and monoclonal antibodies has significant implications:

Currently available commercial MRAS Antibody, FITC conjugated products are predominantly polyclonal antibodies raised in rabbits , offering good versatility across applications but requiring careful validation for specificity.

How can MRAS Antibody, FITC conjugated be used in cancer research studies?

Given that RAS family proteins are oncogenically activated in a significant fraction of tumors , MRAS Antibody, FITC conjugated offers valuable applications in cancer research:

• Expression profiling:

  • Analyze MRAS expression patterns across cancer types and stages

  • Compare with normal tissue counterparts

  • Correlate with patient outcome data

• Signaling pathway analysis:

  • Investigate MRAS activation in relation to MAPK pathway dysregulation

  • Study co-localization with oncogenic signaling components

  • Assess effects of targeted therapies on MRAS localization and activity

• Cytoskeletal reorganization studies:

  • Examine MRAS involvement in cancer cell migration

  • Analyze focal adhesion dynamics in invasive cells

  • Study cytoskeletal changes during epithelial-mesenchymal transition

• Multiplexed approaches:

  • Combine with other cancer markers for comprehensive profiling

  • Use in tissue microarray analysis for high-throughput screening

  • Implement in multiparameter flow cytometry for circulating tumor cells

What considerations are important when using MRAS Antibody, FITC conjugated in tissue microenvironments?

Tissue microenvironments present unique challenges for immunofluorescence studies:

• Tissue-specific optimization:

  • Different tissues require adjusted fixation protocols

  • Antigen retrieval methods should be optimized for each tissue type

  • Blocking protocols need modification for tissues with high endogenous biotin or Fc receptors

• Autofluorescence management:

  • Tissues contain autofluorescent components (lipofuscin, elastin, collagen)

  • Consider spectral imaging to separate FITC signal from autofluorescence

  • Use tissue-specific quenching protocols prior to antibody incubation

• Penetration considerations:

  • Thicker sections require longer incubation times

  • Consider using fragment antibodies for better tissue penetration

  • Optimization of detergent concentration for adequate permeabilization

• Control recommendations:

  • Include tissue-matched negative controls

  • Process comparative tissues in parallel

  • Consider multiplex staining with known markers to establish context