GSS Antibody, FITC conjugated

What is Glutathione Synthetase (GSS) and what is its significance in research?

Glutathione Synthetase (GSS) is a critical enzyme in the glutathione synthesis pathway, catalyzing the second step in glutathione biosynthesis. This 474-amino acid protein (UniProt ID: P48637) has significant implications in neuroscience and signal transduction research . GSS functions to combine glycine with γ-glutamylcysteine to form glutathione, a tripeptide with crucial antioxidant properties that protects cells from oxidative damage and participates in detoxification processes.

Research significance includes:

• Role in oxidative stress response mechanisms

• Implications in neurodegenerative disorders

• Function in cellular detoxification pathways

• Involvement in drug resistance mechanisms in cancer

Detection and quantification of GSS using specific antibodies allows researchers to investigate these pathways in various experimental models.

What distinguishes FITC-conjugated GSS antibodies from other detection methods?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent visualization of GSS without requiring secondary antibody steps. The FITC-conjugated GSS antibodies emit green fluorescence (excitation ~495nm, emission ~519nm) when bound to the target protein . This differs from traditional detection methods like:

FITC-conjugated GSS antibodies are particularly valuable in applications requiring direct visualization including immunofluorescence, immunocytochemistry, immunohistochemistry, and flow cytometry .

How should I store and reconstitute the FITC-conjugated GSS antibody?

Proper storage is critical for maintaining antibody functionality. The FITC-conjugated GSS antibody is typically supplied in lyophilized form containing stabilizers like PBS pH 7.4, 20 mg/ml BSA, 0.02% sodium azide, and 4% trehalose . For optimal results:

• Store the lyophilized antibody at 2-8°C (refrigerated)

• Never freeze the antibody as this can damage the conjugate

• For reconstitution:

  • Add sterile PBS or other recommended buffer

  • Allow complete dissolution (15-20 minutes) at room temperature

  • Avoid vigorous vortexing that may denature the antibody

• Once reconstituted, use within 1 month when stored at 2-8°C

• Protect from prolonged light exposure to prevent photobleaching of the FITC fluorophore

What are the recommended applications for GSS antibody, FITC conjugated?

The polyclonal FITC-conjugated GSS antibody has been validated for several research applications where direct visualization of GSS is required :

• Immunofluorescence (IF): For tissue sections or cell preparations to visualize GSS distribution patterns

• Immunocytochemistry (ICC): For detailed subcellular localization in cultured cells

• Immunohistochemistry (IHC): For analysis of GSS expression in tissue specimens

• Flow cytometry (FACS): For quantitative assessment of GSS expression across cell populations

For each application, proper titration is recommended to determine optimal antibody concentration. Typically, starting dilutions of 1:50 to 1:200 are appropriate for most applications, with further optimization based on signal intensity and background levels.

What sample types can be analyzed with FITC-conjugated GSS antibodies?

When working with GSS antibodies, researchers can analyze various biological samples:

• Fixed cell preparations: Particularly useful for ICC applications

• Tissue sections: For IHC analysis of GSS distribution in tissues

• Flow cytometry samples: For cell-by-cell analysis of GSS expression

• Cell lysates: When using complementary detection methods

For quantitative analysis of GSS protein levels, alternative methods like ELISA may be more appropriate, which can assess GSS in serum, plasma, cell culture supernatant, cell/tissue lysates, and other liquid samples .

How can I design a multicolor immunofluorescence experiment incorporating GSS antibody, FITC conjugated?

Multicolor immunofluorescence experiments require careful planning to avoid spectral overlap. Since the GSS antibody is FITC-conjugated (green emission spectrum), design your panel with the following considerations:

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap with FITC

  • Recommended combinations: FITC + TRITC/Texas Red + DAPI

  • Avoid PE as it has significant spectral overlap with FITC

• Sequential staining approach:

  • Begin with the GSS FITC-conjugated antibody

  • Follow with additional primary antibodies with non-overlapping targets

  • Use fluorophore-conjugated secondary antibodies for non-conjugated primaries

• Controls for multicolor experiments:

  • Single-color controls to establish proper compensation

  • Fluorescence minus one (FMO) controls

  • Isotype controls to assess non-specific binding

• Microscope settings:

  • Use narrow bandpass filters to minimize bleed-through

  • Consider sequential scanning for confocal microscopy

  • Implement spectral unmixing algorithms if available

How does GSS antibody perform in bispecific detection systems?

While the standard GSS antibody is monospecific, researchers interested in multiplex detection might consider bispecific approaches. Building on concepts from bispecific antibody technologies, several strategies can be considered:

• Conventional multicolor approach: Use GSS FITC-conjugated antibody alongside other antibodies with different conjugates

• Sequential dual targeting: Building on bispecific antibody concepts, researchers can employ:

  • Primary staining with GSS FITC-conjugated antibody

  • Secondary staining with another antibody system

  • This approach is conceptually similar to bispecific engagement techniques

• Advanced applications: For researchers familiar with recombinant antibody technologies, adaptation of concepts like Format Chain Exchange technology (FORCE) may eventually allow more sophisticated multiplex detection systems

It's worth noting that true bispecific antibodies require specialized engineering approaches beyond standard conjugation methods. Current bispecific development focuses on therapeutic applications rather than research reagents .

What factors influence epitope accessibility when using GSS antibody, FITC conjugated?

Epitope accessibility is critical for successful staining and can be affected by:

• Fixation effects:

  • Paraformaldehyde cross-linking may mask certain epitopes

  • Methanol fixation denatures proteins and may expose different epitopes

  • The GSS antibody (catalog 33029-05141) recognizes human GSS (1-474 aa) and may have differential accessibility depending on fixation

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval (citrate or EDTA buffer)

  • Enzymatic retrieval (proteinase K or trypsin)

  • Optimization required based on tissue type and fixation method

• Permeabilization factors:

  • Membrane proteins may require gentler permeabilization

  • Intracellular targets like GSS require adequate permeabilization

  • Titrate detergent concentration (Triton X-100, saponin) to optimize signal

• Steric hindrance from FITC conjugation:

  • The FITC moiety may occasionally impact antibody binding

  • If reduced signal is observed, compare with unconjugated primary + FITC-secondary approach

How can I minimize background fluorescence when using GSS antibody, FITC conjugated?

Background fluorescence can significantly impact the signal-to-noise ratio when working with FITC-conjugated antibodies:

• Blocking optimization:

  • Use 3-5% BSA or 5-10% serum from the same species as secondary antibody

  • Consider specialized blockers for highly autofluorescent tissues

  • Extended blocking times (1-2 hours) may improve results

• Antibody dilution:

  • Titrate the FITC-conjugated GSS antibody (typically 1:50 to 1:200)

  • Higher dilutions often decrease background but must balance with signal intensity

• Washing procedures:

  • Increase wash duration and number of washes

  • Use 0.05-0.1% Tween-20 in wash buffer to reduce non-specific binding

  • Consider PBS with higher salt concentration for stringent washing

• Autofluorescence reduction:

  • Treat sections with sodium borohydride or glycine to reduce fixative-induced autofluorescence

  • Use Sudan Black B (0.1-0.3%) to quench lipofuscin autofluorescence

  • Consider spectral unmixing during image acquisition

• Sample preparation:

  • Fresh samples often have less autofluorescence

  • Minimize exposure to light and oxidation during preparation

What controls should be included in experiments using GSS antibody, FITC conjugated?

Proper controls are essential for interpreting results with FITC-conjugated GSS antibodies:

• Positive controls:

  • Cell lines or tissues known to express GSS (based on literature)

  • Recombinant GSS protein as a spike-in control

  • The antibody was generated against human GSS (1-474 aa) expressed in E. coli

• Negative controls:

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration

  • Omission of primary antibody to assess secondary reagent specificity

  • Tissues/cells known to lack GSS expression

• Blocking controls:

  • Pre-incubation of the antibody with recombinant GSS protein

  • This should abolish specific staining if the antibody is selective

• Fluorescence controls:

  • Unstained samples to establish autofluorescence levels

  • Single-color controls if performing multiplex imaging

• Validation controls:

  • Correlation with other detection methods (e.g., ELISA)

  • Comparison with mRNA expression data

  • Western blot to confirm specificity for GSS protein

How does FITC-conjugated antibody detection compare with ELISA methods for GSS?

Both detection platforms offer different advantages depending on research needs:

The ELISA-based detection utilizes a sandwich approach with capture and detection antibodies, offering high specificity and sensitivity . In contrast, the direct FITC-conjugated antibody provides spatial information but may have more variable quantification.

What methodological approaches can improve specificity when working with GSS antibody, FITC conjugated?

To enhance specificity and reduce non-specific binding:

• Titration optimization:

  • Perform a dilution series (1:25 to 1:400) of the antibody

  • Select the dilution that maximizes signal-to-noise ratio

  • The affinity-purified nature of the antibody should provide good specificity at appropriate dilutions

• Blocking enhancements:

  • Use species-matched serum at 5-10%

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Consider dual blocking with both protein (BSA/serum) and non-protein blockers

• Buffer optimization:

  • Increase salt concentration (150-300mM NaCl) to reduce ionic interactions

  • Adjust pH slightly (6.8-7.5) to optimize binding conditions

  • Add low concentrations of detergent (0.05% Tween-20)

• Incubation conditions:

  • Longer incubation at lower temperature (4°C overnight) often improves specificity

  • Compare with standard room temperature incubation (1-2 hours)

• Cross-adsorption consideration:

  • If cross-reactivity is observed, consider additional blocking with unrelated tissues

  • Pre-adsorb the antibody with tissues lacking GSS expression