MDM2 Antibody, FITC conjugated

What are the validated applications for MDM2 antibody, FITC conjugated?

MDM2 antibody, FITC conjugated has been validated for multiple applications in research settings, primarily:

• Western Blotting (WB): Typically used at dilutions of 1:1000

• Immunohistochemistry (IHC): Recommended at dilutions of approximately 1:50

• Immunofluorescence (IF): Effective at dilutions ranging from 1:200 to 1:800

• Flow Cytometry (FCM): Particularly useful due to the FITC conjugation

The antibody's FITC conjugation makes it especially valuable for applications requiring direct fluorescence detection without secondary antibody labeling steps. When using this antibody, note that the excitation/emission wavelengths are approximately 499/515nm with compatibility with 488nm laser lines .

Which species does the MDM2 antibody, FITC conjugated reliably detect?

Based on extensive validation data, the MDM2 antibody, FITC conjugated typically demonstrates reactivity with:

• Human MDM2 protein (primary validated target)

• Mouse MDM2 (with confirmed cross-reactivity)

• Rat MDM2 (with confirmed cross-reactivity)

The reactivity profile depends on the specific antibody clone and manufacturer. Most commercially available antibodies are raised against synthetic peptides from human MDM2, particularly regions within amino acids 100-200 or the specific peptide sequence from positions 374-391 .

What is the typical molecular weight of MDM2 that should be detected in Western blots?

When performing Western blot analysis with MDM2 antibodies, researchers should expect to observe:

• Primary band at approximately 90 kDa (full-length MDM2)

• Secondary band at approximately 50-55 kDa (representing isoform 2 or 3)

According to product specifications, the ~50 kDa band specifically represents isoform 2 or 3 of MDM2. When optimizing Western blot protocols, it's important to consider these multiple isoforms to correctly interpret experimental results .

What controls should be included when using MDM2 antibody, FITC conjugated for immunofluorescence studies?

For rigorous immunofluorescence experiments using MDM2 antibody, FITC conjugated, include the following controls:

• Positive control: Cell lines known to express high levels of MDM2 (e.g., certain cancer cell lines, particularly those with MDM2 amplification)

• Negative control:

  • Isotype control (FITC-conjugated non-specific IgG)

  • Cells with MDM2 knockdown (siRNA or CRISPR)

  • Secondary antibody-only control (if using an unconjugated primary with FITC-secondary)

• Absorption control: Pre-absorb the antibody with recombinant MDM2 protein (0.01 μg/μL) overnight at 4°C followed by centrifugation at 10,000 ×g for 15 min, then use the supernatant. This should significantly reduce the specific signal, as demonstrated in immunofluorescence assays using anti-MDM2 positive sera .

• Counterstain: Include nuclear counterstain (DAPI) to confirm the nuclear localization pattern typical of MDM2, as it is predominantly a nuclear phosphoprotein .

How should sample preparation be optimized for detecting MDM2 using FITC-conjugated antibodies?

Optimal sample preparation for MDM2 detection using FITC-conjugated antibodies requires:

• Fixation:

  • For cells: 4% paraformaldehyde for 15-20 minutes at room temperature

  • For tissues: Formalin-fixed, paraffin-embedded (FFPE) sections

• Permeabilization:

  • 0.1-0.5% Triton X-100 for 5-10 minutes is essential for nuclear antigen access

  • Alternative: 0.1% saponin for more gentle permeabilization

• Antigen retrieval (particularly important for FFPE tissues):

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Or EDTA buffer (pH 9.0) for 15-20 minutes

• Blocking:

  • 5-10% normal serum (matching the species of secondary antibody)

  • 1% BSA in PBS to reduce background fluorescence

• Signal preservation:

  • Mount with anti-fade mounting medium

  • Store slides in the dark at 4°C

  • Image promptly as FITC is susceptible to photobleaching

Remember that MDM2 is normally expressed at low levels in most tissues but is overexpressed in various cancer types, making signal optimization crucial for accurate detection .

How can researchers verify the specificity of MDM2 antibody, FITC conjugated in their experimental system?

To verify MDM2 antibody specificity in your experimental system:

• Molecular validation:

  • Perform Western blot analysis to confirm the antibody detects bands of expected molecular weight (~90 kDa for full-length MDM2 and ~50 kDa for isoforms)

  • Compare results with multiple anti-MDM2 antibodies targeting different epitopes

• Genetic validation:

  • Use MDM2 knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9) cells

  • The specific signal should be significantly reduced or absent in these samples

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunogenic peptide

  • This should block specific binding and reduce or eliminate the signal

• Cellular localization pattern:

  • MDM2 should predominantly show nuclear localization

  • Evaluate if the staining pattern matches known MDM2 distribution

• Biological validation:

  • Test in samples where MDM2 expression is experimentally modulated

  • For example, p53 activation should induce MDM2 expression due to the p53-MDM2 feedback loop .

How can MDM2 antibody, FITC conjugated be used to study the p53-MDM2 regulatory loop in cancer cells?

The p53-MDM2 regulatory loop is a critical pathway in cancer biology that can be studied using MDM2 antibody, FITC conjugated through several sophisticated approaches:

• Dual immunofluorescence analysis:

  • Co-stain with MDM2 antibody (FITC conjugated) and p53 antibody (with a different fluorophore)

  • Quantify co-localization in different cellular compartments

  • This approach reveals the spatial relationship between these proteins across different cell states or treatments

• Drug-induced MDM2 modulation studies:

  • Treat cells with MDM2 inhibitors like Nutlin-3, which disrupts the MDM2-p53 interaction

  • Monitor real-time changes in MDM2 levels and localization with the FITC-conjugated antibody

  • Nutlin-3 has been shown to enhance MDM2 expression while simultaneously increasing HLA-class I and HLA-DR expression

• Cell cycle analysis:

  • Combine MDM2-FITC staining with DNA content analysis

  • This allows correlation of MDM2 expression with specific cell cycle phases

  • Flow cytometry can be used to quantify these relationships

• Stress response dynamics:

  • Subject cells to DNA-damaging agents that activate the p53 pathway

  • Use time-lapse imaging to track changes in MDM2-FITC signal intensity and localization

  • This approach reveals the temporal dynamics of the p53-MDM2 feedback loop

The p53-MDM2 loop functions as a feedback-control mechanism where p53 activation upregulates MDM2, which then inhibits p53 activity and promotes its degradation . This relationship makes it an important target for cancer therapeutics.

What are the methodological considerations when using MDM2 antibody, FITC conjugated for detecting MDM2 in tumor samples?

Detecting MDM2 in tumor samples using FITC-conjugated antibodies requires specific methodological considerations:

• Tissue preparation considerations:

  • Fresh frozen tissues often preserve antigenicity better than FFPE samples

  • If using FFPE, optimize antigen retrieval methods specifically for MDM2

  • Section thickness (4-6 μm optimal) affects antibody penetration

• Signal-to-noise optimization:

  • Autofluorescence is a significant challenge in tumor tissues

  • Pre-treatment with Sudan Black B (0.1-0.3%) can reduce autofluorescence

  • Alternatively, use spectral unmixing during image acquisition

• Heterogeneity assessment:

  • MDM2 expression is often heterogeneous within tumors

  • Systematic sampling of multiple tumor regions is essential

  • Consider tissue microarrays for high-throughput analysis

• Quantification strategies:

  • Develop consistent scoring systems (0-3+ or H-score)

  • Use digital image analysis with appropriate thresholding

  • Report both percentage of positive cells and intensity metrics

• Clinical correlation methods:

  • MDM2 overexpression correlates with poor prognosis in various cancers

  • Document relevant clinicopathological parameters

  • Consider multi-marker panels including p53 status

MDM2 overexpression is detected in a variety of cancers and can result in excessive inactivation of p53, diminishing its tumor suppressor function . Blue fluorescent dyes like FITC are not ideal for detecting low abundance targets due to lower fluorescence and potentially higher non-specific background .

How can MDM2 antibody, FITC conjugated be used to investigate autoimmune responses in systemic lupus erythematosus?

MDM2 antibody, FITC conjugated can be instrumental in investigating the emerging role of MDM2 in systemic lupus erythematosus (SLE) through several methodological approaches:

• Detection of anti-MDM2 autoantibodies:

  • Use purified recombinant MDM2 protein to capture patient autoantibodies

  • Then employ MDM2-FITC conjugated antibody (targeting a different epitope) to detect bound MDM2

  • This two-step approach can confirm the presence of anti-MDM2 autoantibodies

• Cellular distribution analysis:

  • Compare MDM2 expression patterns in immune cells from SLE patients versus healthy controls

  • Quantify nuclear vs. cytoplasmic distribution using confocal microscopy

  • Research shows anti-MDM2 positive sera present a distinct nuclear staining pattern

• Correlative studies with disease markers:

  • Analyze relationships between MDM2 expression and established SLE markers

  • Studies have demonstrated that anti-MDM2 autoantibody was detected at 23.30% prevalence in SLE patients compared to only 4.30% in healthy controls

  • The titer of anti-MDM2 positively correlates with anti-p53 in SLE patients (39.50% prevalence)

• Treatment response monitoring:

  • Track changes in MDM2 expression in response to immunosuppressive therapies

  • Flow cytometry with MDM2-FITC antibody enables quantitative assessment of therapy effects

Research has shown that MDM2 is overexpressed in the MRL-Fas^lpr mice (an animal model of SLE) and correlates with disease progression. This suggests abnormal MDM2 expression may trigger autoimmune responses, making anti-MDM2 a potential new serological marker or therapeutic target in SLE .

How does the FITC conjugation affect the performance of MDM2 antibody compared to unconjugated versions?

FITC conjugation introduces specific performance considerations for MDM2 antibodies:

Key technical considerations when using FITC-conjugated MDM2 antibodies:

• Photobleaching mitigation:

  • Minimize exposure to light during all steps

  • Use anti-fade mounting media containing DABCO or similar compounds

  • Consider using Slow Fade Gold or ProLong Diamond for extended imaging

• Signal optimization:

  • FITC fluorescence is pH-dependent (optimal at slightly alkaline pH)

  • Use PBS or TBS at pH 7.4-8.0 for best results

  • Blue fluorescent dyes like FITC are not recommended for detecting low abundance targets

• Autofluorescence considerations:

  • FITC emission (515nm) overlaps with cellular autofluorescence

  • Consider alternative CF®488A conjugates (Ex/Em: 490/515nm) for improved performance

What are the common challenges in detecting MDM2 using antibody-based methods and how can they be addressed?

Detecting MDM2 using antibody-based methods presents several specific challenges with corresponding solutions:

• Low endogenous expression levels:

  • Challenge: MDM2 is often expressed at low levels in normal cells

  • Solution: Use signal amplification systems (TSA/CARD) or more sensitive detection methods like RNAscope for validation

  • Approach: Pre-treatment with proteasome inhibitors (e.g., MG132 at 2 μM for 12 hours) can increase MDM2 levels by preventing degradation

• Multiple isoform detection:

  • Challenge: MDM2 exists in multiple isoforms (~90 kDa full-length and ~50 kDa isoforms)

  • Solution: Select antibodies targeting conserved epitopes across isoforms

  • Approach: Verify epitope location relative to alternative splicing sites; the specific ~50 kDa band represents isoform 2 or 3

• Cross-reactivity with related proteins:

  • Challenge: MDM2 shares homology with MDMX/MDM4

  • Solution: Validate specificity using knockout/knockdown controls

  • Approach: Confirm specificity using peptide competition assays

• Dynamic regulation by p53:

  • Challenge: MDM2 levels fluctuate due to p53 feedback regulation

  • Solution: Consider p53 status of experimental models

  • Approach: Document p53 status and standardize experimental conditions

• Nuclear localization barriers:

  • Challenge: MDM2 is predominantly nuclear, requiring efficient antibody nuclear penetration

  • Solution: Optimize permeabilization conditions

  • Approach: Extended permeabilization (0.5% Triton X-100 for 15-20 minutes) can improve nuclear antigen access

• Post-translational modifications:

  • Challenge: Phosphorylation affects antibody recognition

  • Solution: Use phospho-specific antibodies when studying MDM2 regulation

  • Approach: Consider antibodies targeting MDM2 phosphorylated at specific sites like Ser166

What are the optimal storage conditions and shelf life for maintaining the activity of MDM2 antibody, FITC conjugated?

Optimal storage and handling of MDM2 antibody, FITC conjugated requires careful attention to preserve both antibody integrity and fluorophore activity:

• Storage temperature:

  • Store at -20°C for long-term storage

  • Avoid repeated freeze/thaw cycles by preparing small aliquots

  • Some manufacturers recommend -80°C storage for extended shelf life

• Buffer composition:

  • Optimal storage buffer typically contains:

    • PBS, pH 7.4

    • 50% glycerol (cryoprotectant)

    • 0.09% sodium azide (preservative)

  • Avoid buffers containing primary amines which can react with FITC

• Light protection:

  • FITC is highly light-sensitive

  • Store in amber tubes or wrap containers in aluminum foil

  • Minimize exposure to light during all handling steps

• Shelf life considerations:

  • Typical shelf life is 12 months when stored properly

  • FITC conjugates generally have shorter shelf lives than unconjugated antibodies

  • Document date of receipt and first use

• Stability indicators:

  • Visible precipitation indicates denaturation

  • Significant decrease in fluorescence intensity suggests FITC degradation

  • Increasing non-specific background may indicate antibody degradation

• Working solution handling:

  • Prepare fresh working dilutions for each experiment

  • Return stock solutions to -20°C promptly after use

  • Working solutions can be stored at 4°C for up to one week but with diminished performance

Proper storage according to manufacturer recommendations is essential as antibody degradation or FITC photobleaching can lead to inconsistent experimental results and false negatives.

How can MDM2 antibody, FITC conjugated be used to study the role of MDM2 in the ubiquitin-proteasome pathway?

MDM2 antibody, FITC conjugated provides powerful methodological approaches to investigate MDM2's function as an E3 ubiquitin ligase in the ubiquitin-proteasome pathway:

• Co-localization with ubiquitinated proteins:

  • Use dual immunofluorescence with MDM2-FITC and anti-ubiquitin antibodies (different fluorophore)

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Track spatial relationships during proteasomal inhibition

• Monitoring MDM2 autoubiquitination:

  • MDM2 undergoes self-ubiquitination as a regulatory mechanism

  • Time-course experiments with proteasome inhibitors (e.g., MG132)

  • Quantify changes in MDM2-FITC intensity as indication of accumulation

• Investigation of E3 ligase activity modulation:

  • The TSG101 protein's Ubc domain interferes with ubiquitination of MDM2

  • Study effects of TSG101 overexpression on MDM2 levels using microscopy

  • TSG101 inhibits MDM2 decay and elevates its steady-state level

• Ubiquitination targets visualization:

  • MDM2 targets p53, Rb, and ribosomal protein L5 for degradation

  • FITC-labeled MDM2 allows tracking of interaction with these targets

  • Study dynamics of MDM2-substrate interactions using live-cell imaging

• His-tagged ubiquitin pulldown assays:

  • Transfect cells with His-tagged ubiquitin (HM-Ub or HM-K48R-Ub)

  • Purify ubiquitinated proteins using Ni-NTA columns

  • Visualize MDM2-containing complexes using the FITC-conjugated antibody

Research has shown that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, inhibits MDM2 decay, elevates its steady-state level, and these events are associated with down-regulation of p53 protein .

What methodological approaches can be used to study MDM2's interaction with p53 using FITC-conjugated MDM2 antibodies?

Studying the critical MDM2-p53 interaction using FITC-conjugated MDM2 antibodies can be accomplished through several methodological approaches:

• Proximity Ligation Assay (PLA):

  • Combine MDM2-FITC antibody with anti-p53 antibody

  • PLA generates fluorescent spots only when proteins are in close proximity (<40nm)

  • Quantify interaction frequency in different cellular compartments or conditions

• FRET analysis:

  • Use MDM2-FITC as donor and p53 labeled with a compatible acceptor fluorophore

  • Measure energy transfer as indication of direct protein-protein interaction

  • Can provide real-time dynamics of interaction in living cells

• Co-immunoprecipitation visualization:

  • Perform p53 immunoprecipitation followed by MDM2-FITC detection

  • Alternatively, use MDM2 pull-down and detect p53

  • Compare interaction under various cellular stresses

• Drug intervention studies:

  • Apply MDM2 inhibitors like Nutlin-3 that disrupt MDM2-p53 interaction

  • Monitor changes in co-localization patterns

  • Targeting the p53-MDM2 interaction with APG115 augments MDM2 in T cells, stabilizing STAT5 and boosting T cell immunity

• Correlation with functional outcomes:

  • Relate MDM2-p53 interaction patterns to apoptotic markers

  • Assess cell cycle distribution in relation to interaction intensity

  • Link interaction dynamics to downstream p53 target gene expression

The p53-MDM2 pathway controls T cell immunity, and targeting this pathway may be therapeutic in cancer patients regardless of tumor p53 status. MDM2 abundance correlates with T cell function and IFNγ-signature in cancer patients .

How can MDM2 antibody, FITC conjugated be utilized in studying MDM2's role in cancer immunotherapy development?

MDM2 antibody, FITC conjugated provides valuable tools for investigating MDM2's emerging role in cancer immunotherapy through several methodological approaches:

• MDM2 peptide vaccine development:

  • MDM2-derived peptide epitopes (e.g., MDM2 32-46) can elicit antigen-specific T cell responses

  • Use FITC-conjugated antibodies to track MDM2 expression in:

    • Antigen-presenting cells loading MDM2 peptides

    • Target tumor cells expressing MDM2

  • Quantify MDM2 expression levels before and after immunotherapy

• T cell reactivity assessment:

  • MDM2-specific CD4+ T cells can directly kill tumor cells via granzyme B

  • Use flow cytometry with MDM2-FITC antibody to:

    • Identify MDM2-high tumor populations

    • Correlate MDM2 expression with susceptibility to T cell killing

  • Combine with T cell activation markers to assess response kinetics

• Combination therapy optimization:

  • MDM2 inhibitors (e.g., Nutlin-3) can enhance antitumor T cell responses by:

    • Increasing MDM2 expression

    • Upregulating HLA-class I and HLA-DR through class II transactivator (CIITA)

  • Use MDM2-FITC antibody to monitor these changes via:

    • Flow cytometry for quantitative assessment

    • Immunofluorescence for spatial distribution analysis

• Biomarker development:

  • MDM2 expression correlates with response to immunotherapy

  • Develop standardized protocols using FITC-conjugated antibodies for:

    • Patient stratification

    • Treatment response monitoring

    • Resistance mechanism identification

Research has shown that MDM2 is expressed in head and neck cancer patients with poor prognosis, and T cells that recognize MDM2 peptides are present in these patients. Using MDM2 peptide vaccines combined with MDM2 inhibitors represents a promising immunotherapeutic strategy .

What are the methodological approaches for using MDM2 antibody, FITC conjugated in studying the role of MDM2 in T cell function and immunity?

Recent research has revealed MDM2's unexpected role in T cell function and anti-tumor immunity. MDM2 antibody, FITC conjugated provides several methodological approaches to investigate this role:

• Competitive binding analysis in T cells:

  • MDM2 competes with c-Cbl for STAT5 binding in T cells

  • Use fluorescence microscopy to visualize:

    • MDM2-FITC distribution

    • STAT5 co-localization

    • c-Cbl displacement

  • This mechanism reduces c-Cbl-mediated STAT5 degradation and enhances STAT5 stability

• T cell functional assessment:

  • MDM2 deficiency in T cells leads to:

    • Accelerated tumor progression

    • Decreased tumor-infiltrating CD8+ T cell survival and function

  • Use MDM2-FITC antibody to sort T cells based on MDM2 expression levels

  • Correlate expression with functional parameters:

    • Cytokine production

    • Cytotoxic activity

    • Proliferative capacity

• Pharmacological intervention studies:

  • APG115 (p53-MDM2 interaction inhibitor) augments MDM2 in T cells

  • This stabilizes STAT5 and boosts T cell immunity

  • Monitor changes in MDM2-FITC signal intensity after treatment

  • Correlate with enhanced anti-tumor activity

• Clinical correlation methodologies:

  • MDM2 abundance correlates with T cell function in cancer patients

  • Develop tissue microarray analysis protocols using MDM2-FITC antibody

  • Correlate expression with:

    • Tumor-infiltrating lymphocyte density

    • IFNγ signature genes

    • Patient outcomes

This research demonstrates that targeting the p53-MDM2 pathway may benefit cancer patients regardless of tumor p53 status by enhancing T cell-mediated anti-tumor immunity .

How can researchers use MDM2 antibody, FITC conjugated to study the relationship between MDM2 and STAT5 in immune regulation?

The emerging relationship between MDM2 and STAT5 in immune regulation can be investigated using MDM2 antibody, FITC conjugated through several sophisticated methodological approaches:

• Competitive binding dynamics visualization:

  • MDM2 competes with c-Cbl for STAT5 binding

  • Use tri-color immunofluorescence to simultaneously visualize:

    • MDM2 (FITC-labeled)

    • STAT5 (different fluorophore)

    • c-Cbl (third fluorophore)

  • Quantify co-localization coefficients under various conditions

• Protein stability assessment protocols:

  • MDM2 enhances STAT5 stability by reducing c-Cbl-mediated degradation

  • Design cycloheximide chase experiments to measure STAT5 half-life

  • Compare STAT5 stability in:

    • MDM2-overexpressing cells

    • MDM2-deficient cells

    • MDM2-inhibitor treated cells

• T cell functional correlation methods:

  • STAT5 is crucial for T cell survival, proliferation and function

  • Sort T cells based on MDM2-FITC expression levels

  • Correlate with STAT5 phosphorylation status

  • Measure functional outputs:

    • IL-2 responsiveness

    • Proliferative capacity

    • Cytotoxic activity

• Dose-response relationship quantification:

  • Transfect cells with varying amounts of MDM2 expression vectors

  • Measure corresponding changes in:

    • STAT5 protein levels

    • STAT5 phosphorylation

    • STAT5 target gene expression

  • Establish mathematical models of the relationship

Research has shown that in tumor-infiltrating CD8+ T cells, MDM2 competes with c-Cbl for STAT5 binding, reducing c-Cbl-mediated STAT5 degradation and enhancing STAT5 stability. This mechanism is critical for T cell-mediated anti-tumor immunity, suggesting potential therapeutic approaches targeting this pathway .

What are the latest methodological approaches for using MDM2 antibody, FITC conjugated in multiplexed imaging systems?

Advanced multiplexed imaging systems offer powerful new opportunities for studying MDM2 biology using FITC-conjugated antibodies through several cutting-edge methodological approaches:

• Cyclic immunofluorescence (CycIF) protocols:

  • Apply MDM2-FITC antibody in first imaging round

  • Capture signal at single-cell resolution

  • Chemically strip or bleach fluorophores

  • Repeat with additional markers (up to 30-40 proteins in same tissue section)

  • Computational alignment and analysis reveals complex relationships between MDM2 and multiple signaling pathways

• Mass cytometry imaging (IMC) integration:

  • While FITC itself isn't compatible with IMC, researchers can:

    • Use serial sections approach

    • Perform FITC-based imaging on one section

    • Use metal-tagged MDM2 antibodies on adjacent section

    • Computationally align images

    • This combines sensitivity of fluorescence with multiplexing capability of IMC

• Spectral unmixing optimization:

  • FITC has spectral overlap with other common fluorophores

  • Latest spectral unmixing algorithms can separate:

    • FITC (MDM2)

    • GFP (if using reporter constructs)

    • Autofluorescence

    • Other spectrally similar fluorophores

  • This enables more complex co-detection panels

• Super-resolution microscopy applications:

  • FITC-conjugated antibodies can be used in techniques like:

    • Structured illumination microscopy (SIM)

    • Stimulated emission depletion (STED)

    • DNA-PAINT (with appropriate DNA-conjugated secondary antibodies)

  • These approaches reveal nanoscale organization of MDM2 relative to binding partners

• Spatial transcriptomics correlation:

  • Combine MDM2-FITC protein detection with:

    • In situ hybridization for MDM2 mRNA

    • Spatial transcriptomics platforms

  • This reveals relationships between:

    • Protein expression

    • Transcriptional regulation

    • Spatial context within tissue architecture