ST5 Antibody, FITC conjugated

What is ST5 Antibody, FITC conjugated and how does it function in research applications?

ST5 Antibody, FITC conjugated is a polyclonal antibody raised in rabbits that specifically recognizes the human Suppression of tumorigenicity 5 protein. The antibody is conjugated with Fluorescein Isothiocyanate (FITC), a fluorescent dye that enables direct visualization of the target protein in various applications. The ST5 antibody targets a specific peptide sequence (amino acids 68-88) of the human ST5 protein .

The ST5 protein itself functions as a guanine nucleotide exchange factor (GEF) that activates RAB9A and RAB9B by promoting the exchange of GDP to GTP, converting inactive GDP-bound Rab proteins into their active GTP-bound form. This protein plays significant roles in cytoskeletal organization and tumorigenicity processes . The FITC conjugation allows researchers to directly detect ST5 protein localization and expression patterns without requiring secondary antibody detection steps.

What are the different isoforms of ST5 and their distinct cellular functions?

The ST5 protein exists in multiple isoforms with distinct biological functions:

• Isoform 1 appears to participate in signaling transduction pathways leading to the activation of MAPK1/ERK2 .

• Isoform 3 demonstrates opposing functionality by potentially blocking ERK2 activation stimulated by ABL1 .

• Isoform 3 may additionally influence cell morphology and growth patterns .

These functional differences between isoforms make ST5 antibodies valuable tools for studying differential signaling pathway activation and cellular phenotypes. Researchers should consider these isoform-specific functions when designing experiments and interpreting results using ST5 antibodies.

What are the optimal conditions for FITC conjugation to antibodies?

The optimal conditions for FITC conjugation to antibodies involve several critical parameters:

• Antibody purity: Using relatively pure IgG obtained by DEAE Sephadex chromatography yields superior conjugation results .

• FITC quality: High-quality FITC reagent is essential for efficient labeling .

• Reaction temperature: Room temperature is optimal for the conjugation reaction .

• pH: A pH of 9.5 provides optimal conditions for the conjugation reaction .

• Protein concentration: An initial protein concentration of 25 mg/ml has been demonstrated to be effective .

• Reaction time: Maximal labeling is typically achieved within 30-60 minutes under these conditions .

Following these parameters closely will help researchers achieve consistent and effective FITC conjugation to ST5 antibodies, ensuring optimal fluorescence intensity while maintaining antibody specificity and activity.

How should researchers determine the optimal FITC-to-antibody ratio?

The fluorescein-to-protein (F/P) ratio is crucial for antibody performance. Typically, between 3 and 6 FITC molecules are conjugated to each antibody molecule . Higher conjugation rates can lead to solubility problems and internal quenching, resulting in reduced brightness .

To determine the optimal ratio, researchers should:

• Set up parallel conjugation reactions with different amounts of FITC while maintaining consistent antibody concentration.

• Compare the resulting conjugates for brightness and background staining.

• Select the conjugation with optimal signal-to-noise ratio for experimental applications.

This optimization process is essential as the FITC conjugation to the antibody may depend on the concentration of antibody in solution. For consistent conjugations, researchers should maintain consistent antibody concentrations across experiments .

What methods are effective for separating optimally labeled antibodies from under- and over-labeled proteins?

After FITC conjugation to antibodies, the reaction mixture contains a heterogeneous population of labeled antibodies. Separation of optimally labeled antibodies from under- and over-labeled proteins can be achieved through gradient DEAE Sephadex chromatography .

This separation technique leverages the fact that FITC conjugation alters the charge characteristics of antibodies, allowing for separation based on the degree of labeling. The process helps isolate antibody fractions with optimal F/P ratios that provide maximum fluorescence intensity while maintaining full immunological activity.

Researchers should validate the separated fractions by:

• Measuring the F/P ratio spectrophotometrically

• Testing antibody specificity via competitive binding assays

• Confirming retained immunological activity through functional assays

How can ST5 Antibody, FITC conjugated be utilized in cancer research and signaling pathway studies?

ST5 Antibody, FITC conjugated serves as a valuable tool in cancer research and signaling pathway studies due to the involvement of ST5 in tumorigenicity and signal transduction processes . Research applications include:

• Direct immunofluorescence staining: Visualizing ST5 protein expression and localization in cancer cell lines and tissue samples.

• Signaling pathway analysis: Investigating the role of ST5 in MAPK/ERK signaling pathways, particularly focusing on how different isoforms differentially regulate ERK activation.

• Cytoskeletal organization studies: Examining the relationship between ST5 and cytoskeletal components in normal versus cancer cells.

• Tumor suppression mechanisms: Exploring how ST5 (originally identified as HeLa tumor suppression 1) functions in suppressing tumor growth and progression.

The FITC conjugation enables direct visualization without secondary detection steps, making it particularly useful for multicolor flow cytometry and microscopy studies where multiple signaling molecules are examined simultaneously.

What are important considerations for storage and handling of FITC-conjugated antibodies to maintain optimal activity?

Proper storage and handling of FITC-conjugated antibodies are crucial for maintaining their activity:

• Storage temperature: Upon receipt, store at -20°C or -80°C for long-term storage (up to 12 months) .

• Avoid freeze-thaw cycles: Repeated freeze-thaw cycles can degrade both the antibody and the fluorophore .

• Short-term storage: For short-term use (up to 1 month), store at 2-8°C under sterile conditions after reconstitution .

• Protection from light: FITC is photosensitive and should be protected from light exposure during storage and handling to prevent photobleaching.

• Buffer conditions: The ST5 Antibody, FITC conjugated is typically stored in a buffer containing 50% glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300 as a preservative .

Adhering to these storage and handling guidelines will help ensure consistent experimental results and extend the usable life of these valuable research reagents.

What factors might contribute to reduced fluorescence intensity when using FITC-conjugated antibodies?

Several factors can contribute to reduced fluorescence intensity with FITC-conjugated antibodies:

• Over-conjugation: Excessive FITC molecules (>6 per antibody) can cause internal quenching, reducing brightness .

• Photobleaching: FITC is susceptible to photobleaching when exposed to light, particularly excitation wavelengths around 488 nm .

• Improper pH: FITC fluorescence is pH-dependent, with optimal fluorescence at slightly alkaline pH (7.5-8.5). Acidic conditions significantly reduce fluorescence intensity.

• Protein denaturation: Improper storage or harsh experimental conditions can denature the antibody, affecting both binding specificity and fluorescence intensity.

• Quenching agents: Certain buffers or sample components (like phenol red) can quench FITC fluorescence.

To mitigate these issues, researchers should optimize conjugation ratios, protect samples from light, use appropriate buffers, and follow proper storage recommendations.

How can researchers enhance FITC signal for low-abundance targets like ST5?

For low-abundance targets like certain ST5 isoforms, researchers can employ several strategies to enhance FITC signal:

• Signal amplification: Using anti-FITC antibodies conjugated to either FITC or other fluorophores with similar excitation and emission spectra can amplify the fluorescence signal .

• Optimized fixation: Different fixation methods can affect epitope accessibility and fluorescence intensity. Testing multiple fixation protocols may improve results.

• Antigen retrieval: For tissue sections or fixed cells, appropriate antigen retrieval methods can enhance antibody binding and subsequent fluorescence signal.

• Reduced background: Optimizing blocking conditions and including appropriate controls can improve signal-to-noise ratio.

• Confocal microscopy: Using confocal microscopy rather than widefield fluorescence can reduce background and improve detection of low-abundance signals.

What experimental controls are essential when working with ST5 Antibody, FITC conjugated?

Rigorous experimental controls are essential for reliable results with ST5 Antibody, FITC conjugated:

• Isotype control: A FITC-conjugated rabbit IgG (non-specific) should be used at the same concentration as the ST5 antibody to assess non-specific binding.

• Blocking/competition control: Pre-incubation of the antibody with excess ST5 immunizing peptide (68-88AA) should abolish specific staining.

• Positive control: Include samples known to express ST5 (based on literature or validated by other methods).

• Negative control: Include samples known not to express ST5 or where ST5 has been knocked down/out.

• Autofluorescence control: Unstained samples to assess natural cellular autofluorescence in the FITC channel.

• Multi-channel controls: When performing multi-color experiments, single-stained controls are necessary to set compensation parameters.

These controls help distinguish specific from non-specific signals and validate experimental findings, particularly important when studying proteins like ST5 that exist in multiple isoforms with different functional roles.

How does the performance of polyclonal ST5 Antibody, FITC conjugated compare with monoclonal alternatives?

While the search results specifically mention polyclonal ST5 Antibody, FITC conjugated , it's important for researchers to understand the comparative advantages and limitations:

Polyclonal ST5 Antibody, FITC conjugated:

• Recognizes multiple epitopes on the ST5 protein, potentially providing stronger signals

• May detect various ST5 isoforms simultaneously

• Useful for applications where high sensitivity is required

• Possible batch-to-batch variation requiring validation

Monoclonal alternatives (theoretical comparison):

• Recognize a single epitope, providing higher specificity

• More consistent between batches

• May be isoform-specific depending on the epitope

• Potentially less sensitive for certain applications

Researchers should select the appropriate antibody type based on their specific experimental needs, considering factors such as required specificity, sensitivity, and consistency across experiments.

What experimental approaches can differentiate between the functions of different ST5 isoforms?

Given that ST5 exists in multiple isoforms with distinct functions , researchers may want to differentiate between them:

• Isoform-specific antibodies: If available, use antibodies targeting isoform-specific regions rather than the shared 68-88AA region.

• Co-localization studies: Combine ST5 antibody with markers for MAPK/ERK pathway components to identify functional associations of different isoforms.

• Genetic approaches: Use isoform-specific siRNA/shRNA to knock down individual isoforms and assess functional consequences.

• Recombinant expression: Express individual isoforms in cell models and assess differential effects on:

  • ERK activation (particularly comparing isoform 1 vs. isoform 3)

  • Cell morphology and growth characteristics

  • Cytoskeletal organization

  • Interactions with RAB9 proteins

• Mass spectrometry: Use quantitative proteomics to identify isoform-specific interaction partners following immunoprecipitation.

These approaches enable researchers to dissect the specific functions of ST5 isoforms in various cellular contexts, providing deeper insights into their roles in normal physiology and disease states.

How should researchers quantify and analyze fluorescence data from ST5 Antibody, FITC conjugated experiments?

Quantification and analysis of fluorescence data from experiments using ST5 Antibody, FITC conjugated require rigorous approaches:

• Flow cytometry analysis:

  • Report median fluorescence intensity (MFI) rather than mean values to minimize the impact of outliers

  • Always subtract background autofluorescence using unstained controls

  • Present data as histograms or dot plots showing population distributions rather than single values

• Microscopy quantification:

  • Use consistent exposure settings across all experimental conditions

  • Quantify multiple fields (minimum 5-10) and cells (>100) per condition

  • Consider colocalization coefficients (Pearson's or Mander's) when assessing ST5 localization with other proteins

• Western blot correlation:

  • When possible, correlate fluorescence intensity with protein quantification by Western blot

  • This helps validate that fluorescence signals accurately represent protein levels

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Report biological replicates (n≥3) rather than technical replicates

  • Include measures of variation (standard deviation or standard error)

Proper quantification ensures reproducible and meaningful interpretation of ST5 localization and expression patterns across experimental conditions.

What challenges exist in interpreting ST5 localization data in relation to its GEF activity?

Interpreting ST5 localization data presents several challenges related to its function as a guanine nucleotide exchange factor (GEF):

• Dynamic interactions: As a GEF that activates RAB9A and RAB9B , ST5 may have transient interactions that are difficult to capture with fixed-cell imaging.

• Activation state: FITC-conjugated antibodies cannot directly distinguish between active and inactive ST5, requiring complementary approaches to assess GEF activity.

• Subcellular compartmentalization: ST5 may relocalize depending on activation state, cell cycle, or signaling events, requiring careful temporal analysis.

• Isoform complexity: Different isoforms may localize to different subcellular compartments or interact with different downstream effectors .

• Context-dependent function: ST5's role in cytoskeletal organization suggests its localization and function may vary with cell type and physiological state.

Researchers should address these challenges by combining fluorescence microscopy with functional assays for GEF activity, co-immunoprecipitation studies, and live-cell imaging where possible to correlate localization with function.

By understanding these complexities, researchers can better interpret ST5 localization data in the context of its GEF activity and downstream signaling effects.

Table: Comparison of ST5 Antibody Applications and Methodological Considerations