GPR107 Antibody, Biotin conjugated

What is GPR107 and why is it significant for research?

GPR107, also known as KIAA1624 or LUSTR1, is a 600 amino acid multi-pass membrane protein belonging to the LU7TM family. It functions as a G protein-coupled receptor with localization primarily to the trans-Golgi network (TGN) . The significance of GPR107 spans multiple biological domains:

GPR107 has been identified as essential for intoxication by bacterial toxins, particularly Pseudomonas aeruginosa exotoxin A (PE), suggesting its crucial role in intracellular trafficking pathways . Additionally, GPR107 is involved in retrograde protein transport mechanisms and may function as a receptor associating with G-proteins to regulate membrane trafficking within cells . Recent research also suggests that GPR107 serves as a candidate receptor for neuronostatin, potentially playing an important role in central control of cardiovascular function .

Moreover, new findings indicate GPR107 expression in both renal medulla and renal cortex, with its deficiency potentially promoting the development of certain kidney conditions, suggesting broader physiological roles than previously understood .

How does a biotin-conjugated GPR107 antibody enhance detection systems?

Biotin conjugation to GPR107 antibodies provides several methodological advantages in research applications:

The biotin-streptavidin system offers one of the strongest non-covalent biological interactions known (Kd ≈ 10^-15 M), enabling highly sensitive detection of GPR107 even when expressed at low levels. This conjugation allows for signal amplification through multiple layers of detection - the biotin tag can bind multiple streptavidin molecules, each potentially carrying reporter enzymes or fluorophores.

For GPR107 detection in complex cellular environments like the Golgi apparatus where it predominantly localizes , biotin conjugation provides enhanced signal-to-noise ratio compared to directly labeled primary antibodies. This is particularly valuable when studying GPR107's involvement in intracellular trafficking pathways, where signal specificity is crucial.

The versatility of biotin conjugation allows the same primary antibody preparation to be used with different detection systems (fluorescent, chemiluminescent, colorimetric) simply by changing the streptavidin conjugate, providing flexibility in experimental design.

What protocols are recommended for Western blot applications?

For optimal Western blot detection of GPR107 using biotin-conjugated antibodies:

Sample Preparation:

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Sonicate briefly to shear genomic DNA

• Centrifuge at 14,000g for 15 minutes at 4°C to remove debris

• Quantify protein concentration using BCA or Bradford assay

Gel Electrophoresis and Transfer:

• Load 20-50 μg of protein per lane on 10% SDS-PAGE gels

• Run gel at 100V until the dye front reaches the bottom

• Transfer to PVDF membrane at 100V for 1 hour in cold transfer buffer

Immunodetection Protocol:

• Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Incubate with biotin-conjugated GPR107 antibody (dilution 1:500-1:2000) overnight at 4°C

• Wash 3 times with TBST, 5 minutes each

• Incubate with streptavidin-HRP (1:5000) for 1 hour at room temperature

• Wash 3 times with TBST, 5 minutes each

• Develop using ECL substrate and image

Expected result: Detection of GPR107 at approximately 67 kDa, corresponding to its molecular weight of 66,990 Da .

How can the specificity of GPR107 antibody detection be validated?

Rigorous validation of biotin-conjugated GPR107 antibody specificity is essential for reliable research outcomes. Multiple complementary approaches should be employed:

Genetic Validation Approaches:

• Test antibody reactivity in GPR107 knockout models, which should show complete absence of signal

• Use GPR107 gene-trap mutant cell lines like those developed in KBM7 cells

• Conduct knockdown experiments using shRNA against GPR107, comparing signal intensity with control shRNA (e.g., shGFP) as described in previous studies

Protein-Based Validation:

• Perform peptide competition assays using the immunizing peptide derived from the internal region of human GPR107

• Validate with recombinant GPR107 protein expressing epitope tags

• Compare signal patterns with alternative GPR107 antibodies recognizing different epitopes

Expression Pattern Consistency:

• Confirm the expected subcellular localization to the Golgi apparatus

• Verify tissue expression patterns match known GPR107 distribution in renal medulla and cortex

A comprehensive validation matrix showing results across these methods provides the strongest evidence for antibody specificity.

What is known about GPR107's role in bacterial toxin trafficking?

GPR107 has emerged as a critical host factor in bacterial toxin trafficking pathways, particularly for Pseudomonas aeruginosa exotoxin A (PE):

A genome-wide haploid genetic screen in KBM7 cells identified GPR107 as essential for PE intoxication . Mechanistically, GPR107 appears to function in the retrograde trafficking pathway that bacterial toxins exploit to reach their intracellular targets. The protein localizes to the trans-Golgi network and undergoes cleavage by furin protease, which was also identified in the genetic screen .

This involvement in toxin trafficking has significant implications for understanding fundamental cellular transport mechanisms. The identification of GPR107 through bacterial toxin screening demonstrates how toxins can serve as molecular tools to discover novel components of cellular trafficking pathways.

Research using biotin-conjugated GPR107 antibodies can illuminate these pathways by:

• Tracking GPR107 redistribution during toxin exposure

• Identifying toxin-GPR107 colocalization through dual-labeling approaches

• Investigating the temporal dynamics of GPR107 processing during toxin trafficking

What methodologies are recommended for studying GPR107-neuronostatin interactions?

Investigating the interaction between GPR107 and neuronostatin, its potential ligand involved in cardiovascular function regulation , requires specialized experimental approaches:

Receptor-Ligand Binding Assays:

• Perform competitive binding assays using biotinylated neuronostatin and membrane preparations containing GPR107

• Utilize surface plasmon resonance (SPR) with purified GPR107 and varying concentrations of neuronostatin to determine binding kinetics

• Employ FRET-based proximity assays using fluorescently-labeled neuronostatin and GPR107

Functional Response Measurements:

• Monitor intracellular calcium mobilization following neuronostatin stimulation in cells expressing GPR107

• Assess G-protein activation using [35S]GTPγS binding assays in GPR107-expressing cell membranes

• Measure downstream signaling pathway activation (MAP kinases, cAMP levels) in response to neuronostatin

Visualization Techniques:

• Perform immunocytochemistry with biotin-conjugated GPR107 antibody to track receptor internalization following neuronostatin exposure

• Use live-cell imaging with fluorescently tagged GPR107 to monitor real-time trafficking in response to neuronostatin

• Apply proximity ligation assays to detect close association between GPR107 and neuronostatin in fixed cells

What are the critical parameters for immunohistochemistry using biotin-conjugated GPR107 antibody?

Successful immunohistochemical detection of GPR107 requires careful attention to several critical parameters:

Tissue Preparation Variables:

• Fixation method: 4% paraformaldehyde is generally preferred over formalin for GPR107 detection

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes optimizes GPR107 signal

• Section thickness: 5-7 μm sections provide optimal resolution for GPR107 localization

Immunostaining Protocol Optimization:

• Block endogenous biotin using a commercial biotin blocking kit

• Use 10% normal serum from the species of the secondary antibody host

• Apply biotin-conjugated GPR107 antibody at 1:100-1:300 dilution

• Incubate overnight at 4°C in a humidified chamber

• Detect with streptavidin-conjugated reporter system (HRP or fluorophore)

Controls and Validation:

• Include negative controls omitting primary antibody

• Use positive control tissues known to express GPR107 (kidney tissue showing renal medulla and cortex expression)

• Consider dual-labeling with Golgi markers to confirm proper subcellular localization

Troubleshooting Common Issues:

• High background: Increase blocking time or concentration, decrease antibody concentration

• Weak or absent signal: Optimize antigen retrieval method, increase antibody concentration

• Non-specific binding: Use more stringent washing, optimize antibody dilution

How can GPR107 detection be optimized in challenging cell types?

Different cell types present unique challenges for GPR107 detection due to varying expression levels, cellular architecture, and potential interfering factors:

Cell-Type Specific Optimization Table:

Detection Enhancement Strategies:

• Employ tyramide signal amplification (TSA) for very low abundance expression

• Use fluorescent anti-biotin secondary antibodies instead of streptavidin for reduced background

• Consider quantum dot-conjugated streptavidin for photostable, bright signals

• Implement spectral unmixing for multi-color experiments to reduce autofluorescence interference

What approaches are recommended for studying GPR107 post-translational modifications?

GPR107 undergoes several post-translational modifications that influence its function, trafficking, and antibody recognition:

Proteolytic Processing:
GPR107 undergoes cleavage by furin protease, which was identified alongside GPR107 in genetic screens for factors involved in toxin trafficking . This processing appears essential for GPR107 function. To study this:

• Use paired antibodies recognizing epitopes on either side of the cleavage site

• Employ pulse-chase experiments with biotin-conjugated antibodies to track the temporal dynamics of processing

• Compare detection patterns in furin-deficient versus wild-type cells

Glycosylation Analysis:
As a transmembrane protein, GPR107 likely undergoes N-linked glycosylation that may affect antibody recognition:

• Treat samples with glycosidases (PNGase F, Endo H) prior to Western blotting to assess glycosylation status

• Use lectin co-staining to characterize glycan modifications

• Compare antibody reactivity between glycosylated and deglycosylated forms

Phosphorylation Studies:

• Combine immunoprecipitation with biotin-conjugated GPR107 antibodies followed by phospho-specific detection methods

• Use phosphatase treatments to determine if antibody epitopes are phosphorylation-sensitive

• Apply mass spectrometry analysis of immunoprecipitated GPR107 to identify phosphorylation sites

How is GPR107 implicated in kidney pathophysiology?

Recent research has revealed important connections between GPR107 and kidney function:

GPR107 is expressed in both the renal medulla and renal cortex, suggesting widespread distribution throughout kidney tissue . Intriguingly, emerging research indicates that GPR107 deficiency may promote the development of certain kidney conditions, potentially including diabetic nephropathy (DN) .

Diabetic nephropathy is characterized by glomerular basement membrane (GBM) thickening, primarily resulting from abnormal accumulation of collagen type IV (COL4) in the extracellular matrix of podocytes . Podocyte endocytosis plays a crucial role in maintaining COL4 balance and GBM integrity, and GPR107 may be involved in these trafficking processes.

Research Approaches Using Biotin-Conjugated GPR107 Antibodies:

• Comparative immunohistochemistry of kidney sections from healthy and diabetic models

• Co-localization studies with endocytic pathway markers in podocytes

• Tracking changes in GPR107 expression and localization during disease progression

• Correlation of GPR107 levels with collagen accumulation and basement membrane thickness

This emerging area represents a promising direction for future research with potential therapeutic implications for kidney diseases.

What are the latest findings on GPR107's involvement in signal transduction pathways?

GPR107 has been implicated in multiple cellular functions including signal transduction and cell growth regulation , with several emerging research directions:

Neuronostatin Signaling:
GPR107 serves as a promising candidate receptor for neuronostatin, potentially playing an important role in the central control of cardiovascular function . This peptide-receptor interaction may activate specific G-protein signaling cascades that influence cardiovascular regulation.

Retrograde Transport Regulation:
As GPR107 localizes to the trans-Golgi network and is involved in retrograde protein transport , it may connect membrane trafficking events to cellular signaling cascades. The protein potentially functions as a receptor that associates with G-proteins to regulate intracellular membrane transport processes .

Research Tools and Approaches:

• Proximity labeling methods combined with biotin-conjugated GPR107 antibodies for purification

• FRET/BRET biosensors to monitor GPR107-G protein interactions in real-time

• Transcriptomic and proteomic profiling of GPR107-deficient versus wild-type cells

• Pharmacological modulation of suspected downstream pathways

How can advanced imaging techniques enhance GPR107 trafficking studies?

Understanding the dynamic trafficking of GPR107 between cellular compartments requires sophisticated imaging approaches:

Super-Resolution Microscopy Applications:

• Stimulated Emission Depletion (STED) microscopy: Resolve GPR107 distribution within Golgi subcompartments with ~30nm resolution

• Single-Molecule Localization Microscopy (PALM/STORM): Track individual GPR107 molecules with nanometer precision

• Structured Illumination Microscopy (SIM): Achieve 100nm resolution to visualize GPR107 trafficking vesicles

Live-Cell Imaging Strategies:

• Pulse-chase experiments with biotin-conjugated antibodies to follow GPR107 trafficking over time

• Photoactivatable or photoconvertible GPR107 fusion proteins to track specific protein populations

• Correlative Light and Electron Microscopy (CLEM) to connect fluorescence observations with ultrastructural context

Quantitative Analysis Methods:

• Single-particle tracking of GPR107-positive vesicles

• Fluorescence Recovery After Photobleaching (FRAP) to measure GPR107 membrane dynamics

• Ratiometric imaging to quantify GPR107 distribution between compartments

These advanced imaging approaches, when combined with biotin-conjugated antibodies that offer exceptional signal-to-noise properties, provide powerful tools for unraveling the complex trafficking behaviors of GPR107.