AGPS Antibody, FITC conjugated

What is AGPS and what cellular functions does it perform?

AGPS (Alkylglycerone Phosphate Synthase) is a 658 amino acid enzyme that plays a critical role in glycerolipid metabolism and ether lipid biosynthesis. It is localized to the inner aspect of the peroxisomal membrane and likely functions as part of a heterotrimeric complex with GNPAT and a modified form of GNPAT. AGPS contains one FAD-binding PCMH-type domain and utilizes FAD as a cofactor in the synthesis of alkyl-glycerone 3-phosphate and a long-chain acid anion from 1-acetyl-glycerone 3-phosphate and long-chain alcohols. Mutations in the AGPS gene result in rhizomelic chondrodysplasia punctata type 3, characterized by vertebral disorders, severe mental retardation, cutaneous lesions, cataracts, and rhizomelic shortening of the humerus and femur .

What is the molecular weight of AGPS protein and how is it detected in western blotting?

AGPS has an expected molecular weight of approximately 73 kDa when detected using western blot analysis. When performing electrophoresis, it is recommended to use a 5-20% SDS-PAGE gel run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours. After transfer to nitrocellulose membrane, the AGPS antibody can detect a specific band at approximately 73 kDa across various human cell lines including HeLa, K562, HepG2, RT4, HEL, CACO-2, and SiHa whole cell lysates .

What are the recommended storage conditions for FITC-conjugated antibodies?

FITC-conjugated antibodies should be stored protected from light to prevent photobleaching of the fluorophore. For short-term storage (up to six months), store at 4°C. For long-term storage, aliquot the antibody and store at -20°C or -80°C protected from light exposure. Repeated freezing and thawing cycles should be avoided as they may result in loss of antibody activity . Some antibodies may be lyophilized and require reconstitution before use, after which they can be stored at 4°C for one month or aliquoted and frozen at -20°C for six months .

What are the considerations for using FITC-conjugated AGPS antibody in multi-color immunofluorescence experiments?

When designing multi-color immunofluorescence experiments using FITC-conjugated AGPS antibody, researchers must consider:

• Spectral overlap: FITC emits in the green spectrum (~520 nm) and may overlap with other fluorophores like PE or TRITC

• Sequential scanning: Use sequential rather than simultaneous scanning in confocal microscopy to minimize bleed-through

• Compensation: Apply appropriate compensation when using flow cytometry for accurate signal separation

• Relative signal strength: Adjust exposure settings for balanced visualization of all fluorophores

• Photobleaching rates: FITC photobleaches relatively quickly compared to other fluorophores, so consider imaging order

These considerations ensure accurate localization and quantification of AGPS in relation to other cellular markers .

What is the relationship between AGPS expression and peroxisomal disorders?

Defects in the gene encoding AGPS result in rhizomelic chondrodysplasia punctata type 3 (RCDP3), a severe peroxisomal disorder. AGPS is essential for ether lipid biosynthesis, particularly plasmalogens, which are critical membrane components especially abundant in nervous tissue, cardiac tissue, and immune cells. In RCDP3, the enzymatic activity of AGPS is compromised, leading to deficient plasmalogen synthesis. This deficiency manifests clinically as vertebral disorders, severe mental retardation, cutaneous lesions, cataracts, and rhizomelic (proximal) shortening of the humerus and femur. Immunofluorescence studies using FITC-conjugated AGPS antibodies can help characterize the subcellular localization patterns of mutant AGPS proteins and correlate these patterns with disease severity and biochemical abnormalities .

How can FITC-conjugated AGPS antibodies contribute to understanding the heterotrimeric complex formation in peroxisomes?

AGPS functions as part of a presumed heterotrimeric complex with GNPAT and modified GNPAT at the peroxisomal membrane. FITC-conjugated AGPS antibodies enable researchers to:

• Visualize the spatial organization of this complex through co-localization studies with other components

• Investigate protein-protein interactions through proximity ligation assays

• Examine complex assembly/disassembly dynamics under different metabolic conditions

• Study the effects of mutations on complex formation using patient-derived cells

• Track complex formation during peroxisomal biogenesis

These approaches provide insights into the molecular mechanisms of ether lipid synthesis and how disruptions in this process contribute to peroxisomal disorders .

What is the recommended protocol for immunofluorescence using FITC-conjugated AGPS antibody?

For optimal immunofluorescence detection of AGPS using FITC-conjugated antibody, follow this protocol:

• Grow cells on coverslips in appropriate growth medium until 50-70% confluent

• Remove growth medium and rinse cells gently with PBS

• Fix cells by adding 1 mL cold methanol (-20°C) and incubate for 5-10 minutes at room temperature

• Remove methanol and rinse 2 × 5 minutes with PBS

• Permeabilize cells if needed (depending on fixation method)

• Add 2 mL blocking solution (PBS containing 10% fetal bovine serum) and incubate for 20 minutes at room temperature

• Remove blocking solution and add 1 mL of PBS/10% FBS containing FITC-conjugated AGPS antibody at a 1:500 dilution

• Incubate for 1 hour at room temperature in the dark

• Wash cells 2 × 5 minutes with PBS

• Mount coverslips on slides using appropriate mounting medium

• Observe cells with a fluorescence microscope equipped with a FITC filter

This protocol minimizes non-specific binding while maximizing specific signal detection of AGPS protein .

What are the recommended dilutions for different applications of FITC-conjugated AGPS antibody?

These recommendations serve as starting points, and researchers should empirically determine the optimal dilution for their specific experimental conditions and cell types .

What controls should be included when using FITC-conjugated AGPS antibody?

To ensure experimental rigor when using FITC-conjugated AGPS antibody, include the following controls:

• Negative control: Unstained cells to assess autofluorescence

• Isotype control: FITC-conjugated isotype-matched immunoglobulin (rabbit IgG-FITC) to evaluate non-specific binding

• Blocking control: Pre-incubation of antibody with recombinant AGPS protein to confirm specificity

• Positive control: Cells known to express AGPS (e.g., HeLa, K562, HepG2 cell lines) to verify staining pattern

• Secondary antibody control (for indirect methods): Cells incubated with secondary antibody only

• Knockdown/knockout control: Cells with AGPS expression reduced or eliminated to validate antibody specificity

These controls allow researchers to confidently interpret their results and troubleshoot any unexpected outcomes .

How should samples be fixed and permeabilized for optimal AGPS detection?

The proper fixation and permeabilization of samples are critical for accurate detection of AGPS, which is localized to the peroxisomal membrane. Consider the following recommendations:

• Fixation options:

  • Cold methanol (-20°C) for 5-10 minutes (preferred for peroxisomal proteins)

  • 4% paraformaldehyde for 15-20 minutes followed by permeabilization

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) for paraffin-embedded tissues

• Permeabilization (if using paraformaldehyde):

  • 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

  • 0.1-0.5% saponin in PBS (gentler alternative)

  • 100% cold acetone (-20°C) for 5 minutes

The optimal method may vary depending on cell type and specific experimental conditions. For peroxisomal membrane proteins like AGPS, methanol fixation often provides good results as it both fixes and permeabilizes simultaneously, preserving the antigen accessibility .

What are common causes of weak or no signal when using FITC-conjugated AGPS antibody?

When troubleshooting weak or absent signals with FITC-conjugated AGPS antibody, consider these potential issues and solutions:

Systematic evaluation of these factors can help identify and address the source of signal problems .

How can researchers reduce background fluorescence when using FITC-conjugated antibodies?

High background fluorescence is a common challenge when using FITC-conjugated antibodies. To reduce background and improve signal-to-noise ratio:

• Optimize blocking:

  • Increase blocking time to 30-60 minutes

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Use 10% FBS or 5% BSA in PBS as blocking buffer

• Antibody dilution:

  • Titrate the antibody to find the maximal dilution that gives detectable signal

  • Use more dilute antibody solution if background is high

• Washing steps:

  • Increase number and duration of washes

  • Add 0.05-0.1% Tween-20 to wash buffer

  • Perform washes with gentle agitation

• Sample preparation:

  • Ensure complete fixation to reduce autofluorescence

  • Include glycine treatment (100mM, 10 min) after fixation to quench free aldehydes

  • Consider treatments to reduce endogenous fluorescence

• Imaging settings:

  • Adjust gain and offset settings on microscope

  • Use confocal microscopy to reduce out-of-focus fluorescence

  • Implement deconvolution algorithms to improve signal clarity

What approaches can address photobleaching issues with FITC during imaging?

FITC is particularly prone to photobleaching, which can complicate imaging and quantification. To address photobleaching issues:

• Use anti-fade mounting media containing:

  • p-Phenylenediamine

  • ProLong Gold or similar commercial products

  • DABCO (1,4-diazabicyclo[2.2.2]octane)

• Modify imaging approach:

  • Minimize exposure time during image acquisition

  • Reduce excitation light intensity

  • Capture FITC channel first in multi-channel experiments

  • Use widefield microscopy before switching to confocal for detailed imaging

  • Consider time-lapse imaging with reduced sampling frequency

• Sample preparation:

  • Image samples promptly after staining

  • Store slides in the dark at 4°C if imaging must be delayed

  • Consider oxygen-scavenging systems in mounting media

• Alternative approaches:

  • Use computational methods to correct for photobleaching

  • Consider more photostable green fluorophores (Alexa Fluor 488, DyLight 488) for critical experiments

How can researchers validate the specificity of AGPS antibody staining?

Validating the specificity of AGPS antibody staining is crucial for reliable research outcomes. Implement these validation strategies:

• Genetic approaches:

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

  • Compare staining patterns between wild-type and AGPS-deficient samples

  • Rescue experiments with AGPS re-expression

• Biochemical validation:

  • Peptide competition assays to block specific binding

  • Comparison with multiple antibodies against different AGPS epitopes

  • Western blot validation showing a single band at 73 kDa

• Subcellular localization:

  • Co-staining with peroxisomal markers (PEX14, catalase)

  • Confirm peroxisomal membrane localization pattern

  • Examine alterations in localization under conditions known to affect peroxisomes

• Disease model validation:

  • Compare staining patterns in cells from patients with RCDP3

  • Correlate staining intensity with biochemical measures of AGPS activity

  • Examine staining pattern changes in response to treatments affecting peroxisomal function

What emerging applications utilize FITC-conjugated AGPS antibodies in current research?

FITC-conjugated AGPS antibodies are increasingly being utilized in cutting-edge research applications that extend beyond basic localization studies:

• High-content screening approaches to identify modulators of peroxisomal function

• Live-cell imaging to track peroxisomal dynamics in real-time using cell-permeable FITC-conjugated antibody fragments

• Super-resolution microscopy (STORM, PALM) to examine the nanoscale organization of AGPS within the peroxisomal membrane

• Correlative light and electron microscopy (CLEM) to connect AGPS localization with ultrastructural features

• Tissue microarray analysis to profile AGPS expression across different tissues and disease states

These applications highlight the versatility of FITC-conjugated AGPS antibodies in advancing our understanding of peroxisomal biology and ether lipid metabolism in health and disease .

How can researchers optimize experimental design when studying AGPS in complex biological systems?

When designing experiments to study AGPS in complex biological systems, researchers should consider:

• Experimental controls:

  • Include tissue-specific positive and negative controls

  • Use appropriate isotype controls for immunofluorescence

  • Incorporate genetic controls (knockdown/knockout) when possible

• Technical considerations:

  • Use recommended antibody dilutions (1:50-200 for IF/IHC-P)

  • Optimize fixation and permeabilization for peroxisomal proteins

  • Consider epitope accessibility in different sample preparations

• Biological variables:

  • Account for cell type-specific variations in AGPS expression

  • Consider metabolic state effects on peroxisomal function

  • Evaluate disease-specific alterations in AGPS localization or expression

• Complementary approaches:

  • Combine immunofluorescence with functional assays of ether lipid synthesis

  • Correlate AGPS protein levels with enzymatic activity measurements

  • Integrate omics approaches (proteomics, lipidomics) with localization studies