GGA3 Antibody, FITC conjugated

What is GGA3 and why is it important in cellular research?

GGA3 (Golgi-localized, γ-ear-containing ARF-binding protein 3) belongs to the GGA family of ubiquitous coat proteins that facilitate trafficking of soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes. This trafficking occurs through interactions with TGN-sorting receptors, ARF (ADP-ribosylation factor), and clathrin . GGA3 has a modular structure with an N-terminal VHS (VPS27, Hrs and STAM) domain followed by a GAT (GGA and Tom1) domain, a connecting hinge segment, and a C-terminal GAE (γ-Adaptin ear) domain .

The human GGA3 gene maps to chromosome 17 and encodes a 723 amino acid protein that shares 46% sequence identity with GGA1 and 38% with GGA2 . GGA3 is particularly important because it binds ubiquitinated proteins and membrane cargo molecules with cytosolic acidic cluster-dileucine (AC-LL) motifs . Recent research has revealed GGA3's critical role in neuronal protein transport and its potential implications in neurodegenerative diseases like Alzheimer's .

What are the recommended applications for GGA3 antibody, FITC conjugated?

GGA3 antibody, FITC conjugated is primarily used for immunofluorescence applications. Based on manufacturer recommendations, the optimal dilution for immunofluorescence on paraffin-embedded tissues (IHC-P) is 1:50-200 . The FITC conjugation enables direct fluorescent detection without requiring secondary antibodies, which can be advantageous in multi-labeling experiments.

Other potential applications include flow cytometry for intracellular detection , although this would require optimization of permeabilization protocols to access the intracellular GGA3 protein. The antibody can also be used in colocalization studies to examine GGA3's relationship with other trafficking proteins.

What controls should be included when using GGA3 antibody, FITC conjugated?

When using GGA3 antibody, FITC conjugated, several controls are essential:

• Negative controls: Include samples where the primary antibody is omitted but all other steps are identical, to assess background fluorescence.

• Positive controls: Use tissues or cell lines known to express GGA3, such as HEK293 cells which have been demonstrated to express endogenous GGA3 .

• Knockdown controls: When available, include GGA3 siRNA-treated samples to confirm specificity. The search results mention specific siRNA sequences targeting human GGA3 (1703 TGTGACAGCCTACGATAAA 1721) that have been successfully used .

• Isotype controls: Include a non-specific rabbit IgG FITC-conjugated antibody at the same concentration to control for non-specific binding.

• Subcellular localization validation: Compare staining patterns with expected GGA3 localization in the Golgi and endosomal compartments.

How can GGA3 antibody be used to investigate neuronal trafficking mechanisms?

GGA3 antibody, FITC conjugated, is particularly valuable for studying specialized neuronal trafficking mechanisms. Recent research has revealed that GGA3 is distributed in both dendrites and axons of hippocampal neurons . To effectively study these compartmentalized distributions, researchers can employ microfluidic chamber systems that separate axonal compartments from cell bodies.

A methodological approach demonstrated in the literature involves:

• Culturing primary neurons in microfluidic chambers to physically separate axons from cell bodies

• Detecting endogenous GGA3 in both compartments using Western blot analysis

• Confirming axonal specificity using markers like MAP2B (dendrite-specific) and TubβIII (present in both dendrites and axons)

This approach revealed an axonal/cell side densitometry ratio of 0.10±0.02 (n=4) for endogenous GGA3 , providing quantitative data for GGA3 distribution in neurons. When using FITC-conjugated GGA3 antibody for such studies, researchers should optimize fixation and permeabilization protocols to preserve axonal structures while ensuring antibody access.

What methodologies are recommended for studying GGA3 interaction with G protein-coupled receptors?

GGA3 has been shown to interact with G protein-coupled receptors (GPCRs), particularly α2B-adrenergic receptor (α2B-AR), modulating their trafficking and signaling . To investigate these interactions using GGA3 antibody, FITC conjugated, researchers should consider the following methodological approaches:

• Co-immunoprecipitation (co-IP) studies:

  • Transfect cells with the GPCR of interest (e.g., α2B-AR)

  • Lyse cells in buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 1% protease inhibitors

  • Pre-clear lysates with protein G beads

  • Immunoprecipitate with antibodies against the GPCR

  • Detect GGA3 in immunoprecipitates by Western blotting

• GST fusion protein pulldown assays:

  • Generate GST fusion proteins of GPCR intracellular domains (particularly intracellular loops)

  • Perform pulldown assays with cell lysates expressing GGA3

  • Analyze interaction sites, noting that for α2B-AR, the triple Arg motif in the third intracellular loop interacts with the acidic motif EDWE in the VHS domain of GGA3

• Fluorescence colocalization studies:

  • Use FITC-conjugated GGA3 antibody alongside a differently-labeled GPCR

  • Perform confocal microscopy focusing on trafficking compartments

  • Analyze colocalization quantitatively using appropriate image analysis software

How can GGA3 antibody be used to study Alzheimer's disease mechanisms?

GGA3 has emerged as an important player in Alzheimer's disease (AD) pathogenesis. GGA3 loss of function due to genetic deletion or rare variants has been associated with late-onset AD . Specifically, GGA3 regulates the trafficking of BACE1 (β-site APP cleaving enzyme 1), a key enzyme in amyloid-β production.

To investigate GGA3's role in AD using FITC-conjugated GGA3 antibody, researchers should consider:

• Quantitative immunohistochemistry in brain tissue:

  • Compare GGA3 levels in hippocampal regions between AD models and controls

  • Focus particularly on the CA3 hippocampal mossy fibers, where BACE1 accumulation has been observed in GGA3 knockout mice (44% increase in BACE1 in GGA3−/− mice compared to wild-type)

  • Use standardized image acquisition settings and quantification methods

• Axonal pathology investigation:

  • Examine axonal swellings where BACE1 accumulates in the absence of GGA3

  • Determine if GGA3 levels inversely correlate with BACE1 levels and axonal pathology

  • Study these parameters at early disease stages, before amyloid plaque formation

• Analysis of GGA3 variants:

  • Investigate the effect of GGA3 variants (such as Ins545T) on BACE1 trafficking

  • Use rescue experiments in GGA3−/− neurons to assess variant functionality

What are the optimal fixation and permeabilization conditions for detecting GGA3 in different cellular compartments?

Optimizing fixation and permeabilization is crucial for accurate detection of GGA3 in different cellular compartments:

• Standard formaldehyde fixation:

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

  • This preserves general cellular morphology but may not provide optimal access to all GGA3 pools

• Methanol fixation:

  • 100% methanol for 10 minutes at -20°C

  • More effective for revealing Golgi-associated GGA3 pools

• Permeabilization options:

  • 0.1-0.2% Triton X-100 for 5-10 minutes for general permeabilization

  • 0.05% saponin for more gentle permeabilization that better preserves membrane structures

  • Include permeabilization agent in all antibody incubation buffers when using saponin

• Antigen retrieval for tissue sections:

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

  • Critical for detecting GGA3 in paraffin-embedded tissue sections

How can signal-to-noise ratio be optimized when using GGA3 antibody, FITC conjugated?

FITC-conjugated antibodies can sometimes present challenges with signal-to-noise ratio. To optimize results:

• Blocking optimization:

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

  • Add 0.1-0.3% Triton X-100 to blocking buffer to reduce non-specific binding

  • Consider adding 1% BSA to reduce background

• Antibody concentration:

  • Titrate antibody carefully within the recommended range (1:50-200)

  • Lower concentrations may reduce background but require longer exposure times

• Autofluorescence reduction:

  • Treat sections with 0.1% Sudan Black B in 70% ethanol for 5-10 minutes

  • Use specialized commercial reagents designed to reduce tissue autofluorescence

  • Include a spectral unmixing step during image acquisition if using confocal microscopy

• Storage considerations:

  • Store the FITC-conjugated antibody at -20°C or -80°C, avoiding repeated freeze-thaw cycles

  • Protect from light during storage and all incubation steps

What approaches can be used to study the dynamics of GGA3 trafficking in live cells?

While FITC-conjugated antibodies are primarily used for fixed samples, researchers interested in GGA3 dynamics should consider:

• Complementary approaches with fluorescent protein fusions:

  • Use GFP-GGA3 constructs for live cell imaging

  • Design experiments that combine fixed-cell antibody staining with live-cell dynamics

  • Consider photoactivatable or photoconvertible GGA3 fusions for pulse-chase experiments

• Antibody validation strategy:

  • Use the FITC-conjugated GGA3 antibody to validate localization patterns observed with GFP-GGA3

  • Compare endogenous protein detection (antibody) with exogenous expression (GFP fusion)

  • This approach has confirmed that exogenous GFP-GGA3 recapitulates the distribution pattern of endogenous GGA3 in neurons

• FRAP (Fluorescence Recovery After Photobleaching) analysis:

  • Use GFP-GGA3 alongside markers labeled with spectrally distinct fluorophores

  • Determine dynamics of GGA3 in different cellular compartments

  • Correlate findings with fixed-cell antibody staining patterns

How can GGA3 antibody be used in multiplexed immunolabeling experiments?

For complex trafficking studies, multiplexed labeling is often necessary:

• Fluorophore selection for multiplexing:

  • Pair FITC-conjugated GGA3 antibody with antibodies conjugated to spectrally distinct fluorophores

  • Consider using red fluorophores (e.g., Cy3, Alexa Fluor 594) for cargo proteins

  • Use far-red fluorophores (e.g., Cy5, Alexa Fluor 647) for compartment markers

• Sequential immunolabeling protocol:

  • Apply FITC-conjugated GGA3 antibody first

  • Block with excess rabbit IgG to prevent cross-reactivity

  • Apply additional primary antibodies from different host species

  • Use secondary antibodies with minimal cross-reactivity

• Analysis of protein-protein interactions:

  • Combine with proximity ligation assay (PLA) for detecting interactions

  • Validate interactions identified with co-IP using the FITC-conjugated antibody for detection

  • Apply appropriate controls for each additional antibody in the multiplexed panel

How should colocalization between GGA3 and trafficking markers be quantified?

When analyzing colocalization data from experiments using GGA3 antibody, FITC conjugated:

What considerations are important when interpreting GGA3 levels in disease models?

When using GGA3 antibody to study disease models, particularly neurodegenerative disorders:

• Expression level analysis:

  • Compare GGA3 levels in affected vs. unaffected regions

  • Note that GGA3 is decreased and inversely correlated with BACE1 in the temporal cortices of AD patients

  • Consider analysis by Western blot alongside immunofluorescence for quantitative assessment

• Temporal considerations:

  • Examine multiple time points in disease progression

  • GGA3 deletion exacerbates axonal pathology and triggers BACE1 accumulation in axonal swellings prior to senile plaque formation in mouse models

• Correlation with pathological markers:

  • Analyze relationship between GGA3 levels and established disease markers

  • Consider the relationship between GGA3, BACE1, and amyloid-β pathology

  • Evaluate whether GGA3 changes precede or follow other pathological events