Myc Antibody, FITC conjugated

What is the difference between c-Myc protein antibodies and Myc tag antibodies?

The distinction between these antibody types is fundamental to experimental design. c-Myc antibodies (like clone Y69) recognize the endogenous c-Myc protein, a transcription factor involved in growth-related gene activation. In contrast, Myc tag antibodies (such as clone 9E10) detect the synthetic epitope tag derived from c-Myc's C-terminal region, specifically the amino acid sequence EQKLISEEDL, which is engineered into recombinant proteins .

The c-Myc protein functions as a transcription factor that binds to the core DNA sequence 5'-CAC[GA]TG-3' and activates growth-related genes. It's involved in multiple cellular processes including VEGFA promotion, somatic reprogramming, and embryonic stem cell self-renewal . When designing experiments, researchers should carefully select antibodies that specifically target either the endogenous protein or the tag, depending on experimental goals.

What applications are most suitable for FITC-conjugated Myc antibodies?

FITC-conjugated Myc antibodies excel in several applications:

• Flow Cytometry: Particularly effective for both intracellular and extracellular staining, depending on the protein's expression pattern. Recommended working dilutions typically range from 1-5 μg/mL .

• Immunofluorescence/Immunocytochemistry: Enables direct visualization of tagged proteins within cells without requiring secondary antibody incubation, streamlining experimental workflows .

• CyTOF Applications: Some FITC-conjugated antibodies are validated for mass cytometry applications, allowing multiplexed protein analysis .

For flow cytometry applications specifically, researchers should prepare single-cell suspensions (potentially using TrypLE cell dissociation enzyme for adherent cells), incubate with the FITC-conjugated antibody at appropriate dilutions (typically 1:100-1:200), and analyze using appropriate laser excitation (490nm) and emission (525nm) settings .

How should FITC-conjugated Myc antibodies be stored to maintain optimal performance?

Proper storage significantly impacts antibody performance and shelf life:

• Temperature: Store at 2-8°C in the dark. Never freeze FITC-conjugated antibodies as this can cause fluorophore degradation and reduced signal intensity .

• Light Exposure: Minimize exposure to light during storage and handling to prevent photobleaching of the FITC fluorophore .

• Buffer Conditions: These antibodies are typically supplied in phosphate buffered saline (PBS, pH 7.4) with preservatives such as 0.09-0.15% sodium azide .

• Shelf Life: Most manufacturers guarantee performance for 1 year from the date of receipt when stored properly .

• Aliquoting: For frequent users, creating small, single-use aliquots minimizes freeze-thaw cycles and light exposure.

What controls should be included when using FITC-conjugated Myc antibodies?

Robust experimental design requires appropriate controls:

For flow cytometry specifically, researchers demonstrated proper control implementation by comparing H9 embryonic stem cells stained with c-Myc antibody against mouse IgG controls, which enabled clear differentiation between specific signal and background autofluorescence .

How can I optimize fixation and permeabilization protocols for intracellular Myc tag detection?

Optimization strategies vary by application:

For immunofluorescence microscopy:

• Fix cells in 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 15 minutes

• Block with appropriate buffer (often PBS with 5% normal serum)

• Incubate with FITC-conjugated Myc antibody at manufacturer-recommended dilution (typically 1:100-1:200)

• Counterstain nuclei with DAPI if desired

For flow cytometry (intracellular staining):

• Create single-cell suspensions using appropriate dissociation methods (e.g., TrypLE for 5 minutes at 37°C for embryonic stem cells)

• Fix cells in suspension with 4% paraformaldehyde for 10-15 minutes

• Permeabilize with saponin-based buffer or 0.1% Triton X-100

• Incubate with antibody at 1-5 μg/mL concentration

• Analyze promptly or store briefly in FACS buffer with paraformaldehyde (typically 10 μL of 4% PFA in 500 μL buffer)

How does the position of a Myc tag affect protein structure and function?

The position of the Myc tag can significantly impact protein behavior:

Recent molecular dynamics simulation research on FMC63-based anti-CD19 single-chain variable fragments (scFvs) revealed that:

This research suggests that while the Myc tag is often described as minimally disruptive, its position should be carefully considered based on the protein's structural features and intended application. When designing constructs, researchers should evaluate whether N- or C-terminal tagging would less likely interfere with functional domains .

What are the considerations for multiplexing FITC-conjugated Myc antibodies with other fluorophores?

Effective multiplexing requires careful planning:

• Spectral Properties: FITC has excitation/emission peaks at 490nm/525nm. When designing panels, select fluorophores with minimal spectral overlap such as PE (565nm/578nm) or APC (650nm/660nm) .

• Compensation Controls: For each fluorophore in the panel, prepare single-stained controls to establish compensation matrices. This is particularly important for flow cytometry applications.

• Brightness Hierarchy: Match fluorophore brightness to target abundance. Since FITC has moderate brightness, it's best suited for moderately expressed targets rather than rare or dimly expressed proteins.

• Fixation Effects: Some fluorophores are more sensitive to fixation than others. When multiplexing, standardize fixation protocols to maintain consistent signal across all channels.

• Sequential Staining: For complex panels, consider sequential rather than simultaneous staining to minimize potential antibody interactions.

How can I troubleshoot weak or absent signal when using FITC-conjugated Myc antibodies?

Systematic troubleshooting approach:

• Antibody Integrity: Ensure the antibody hasn't been exposed to excessive light or inappropriate storage conditions. FITC is particularly sensitive to photobleaching .

• Expression Level Verification: Confirm Myc-tagged protein expression using alternative methods such as Western blot with HRP-conjugated Myc antibodies .

• Epitope Accessibility: The Myc tag may be obscured by protein folding or interactions. Try different fixation/permeabilization methods to improve accessibility.

• Titration: Optimize antibody concentration by testing a range of dilutions (typically 1-10 μg/mL) to find the optimal signal-to-noise ratio .

• Signal Amplification: For very low expression levels, consider alternative detection strategies such as biotin-streptavidin systems or switching to brighter fluorophores like PE or Alexa Fluor 488.

• Protein Localization: Verify that your detection method is appropriate for the subcellular localization of your tagged protein. Membrane proteins may require different approaches than nuclear proteins.

What are the best practices for using FITC-conjugated Myc antibodies in CAR-T cell research?

CAR-T cell applications require specific considerations:

• Tag Position Effects: Recent research shows that N-terminal Myc tags can negatively impact CAR-T cell antitumor activity. Molecular dynamics simulations reveal that tags near complementarity-determining regions (CDRs) can cause steric hindrance that interferes with target binding .

• Detection Strategy: For CAR expression verification, researchers typically:

  • Create single-cell suspensions using TrypLE treatment (5 minutes at 37°C)

  • Incubate with FITC-conjugated Myc antibody (1:100 dilution) for 1 hour on ice

  • Wash with FACS buffer (PBS + 5% fetal calf serum)

  • Fix in 4% paraformaldehyde before analysis

• Functional Validation: Since tag position can affect function, correlate CAR expression levels (as measured by FITC intensity) with functional readouts such as cytokine production and cytotoxicity assays.

• Alternative Approaches: When tag interference is a concern, consider using epitope-specific antibodies that directly recognize the CAR's scFv portion rather than relying on tags .

How can I quantitatively assess epitope accessibility and binding efficiency of Myc-tagged proteins?

Quantitative assessment methodologies:

• Flow Cytometry Titration: Perform antibody titration experiments to determine optimal concentration. Plot mean fluorescence intensity against antibody concentration to identify saturation points and calculate the effective binding constant (Kd) .

• Competitive Binding Assays: Measure displacement of FITC-conjugated antibody by unlabeled Myc peptide to assess binding specificity and affinity.

• Intermolecular Interaction Potential (IIP) Analysis: As described in recent research, this computational approach can predict how tag position affects interaction with target proteins and reveal potential steric hindrances .

• Signal-to-Noise Ratio Calculation: Compare median fluorescence intensity of Myc-positive samples to isotype controls using the formula:
  $\text{SNR} = \frac{\text{MFI}_{\text{sample}} - \text{MFI}_{\text{background}}}{\text{SD}_{\text{background}}}$

• Time-Course Binding Studies: Assess association and dissociation rates by measuring fluorescence at different time points after antibody addition and washing.

What purification methods are used for FITC-conjugated antibodies and how might this affect performance?

Understanding production methods helps interpret performance:

• Conjugation Chemistry: FITC reacts with primary amines on antibodies (typically lysine residues). The antibody-to-FITC ratio affects brightness and can vary between manufacturers .

• Purification Approaches:

  • Size-exclusion chromatography removes unconjugated FITC and any aggregated antibody

  • Protein A/G purification removes non-antibody proteins prior to conjugation

  • Most commercial preparations undergo both affinity purification and size-exclusion steps

• Quality Control Metrics:

  • Fluorophore-to-protein ratio (typically 3-7 FITC molecules per antibody for optimal performance)

  • Percent free dye (should be <5%)

  • Antibody recovery post-conjugation (typically 60-80%)

  • Retention of antigen binding assessed by comparing conjugated vs unconjugated forms

• Impact on Applications: Purification method affects:

  • Background fluorescence (insufficient removal of free FITC increases background)

  • Signal intensity (over-conjugation can cause fluorescence quenching or alter binding)

  • Batch-to-batch reproducibility (standardized methods produce more consistent results)

For optimal results, researchers should select antibodies purified using size-exclusion chromatography that specifically removes unconjugated fluorophore, as this reduces background signal in imaging and flow cytometry applications .

How are FITC-conjugated Myc antibodies being utilized in emerging single-cell analysis technologies?

Recent adaptations for cutting-edge technologies:

• CyTOF Applications: Some FITC-conjugated Myc antibodies are now validated for mass cytometry, enabling incorporation into high-dimensional panels for comprehensive cellular phenotyping .

• Single-Cell RNA-Protein Co-Detection: Methods combining transcriptomics with protein detection often use Myc-tagged proteins visualized with FITC-conjugated antibodies to correlate expression with transcriptional profiles.

• Live-Cell Imaging: While traditional FITC applications involve fixed cells, newer membrane-permeable variants enable tracking of Myc-tagged proteins in living cells over time.

• Microfluidic Systems: FITC-conjugated antibodies are compatible with microfluidic-based cell sorting and analysis platforms, enabling high-throughput screening of cells expressing Myc-tagged proteins.

• Super-Resolution Microscopy: Though FITC isn't optimal for super-resolution techniques due to photobleaching concerns, specialized variants with improved photostability are emerging for nanoscale visualization of Myc-tagged structures.