GPR123 Antibody

What is GPR123 and what is its biological significance?

GPR123 (G protein-coupled receptor 123), also known as ADGRA1 (adhesion G protein-coupled receptor A1), is a 1,279 amino acid multi-pass membrane protein belonging to the G-protein coupled receptor 2 family and LN-TM7 subfamily . It functions as an orphan receptor, meaning its endogenous ligand remains unidentified.

The GPR123 protein exists as two alternatively spliced isoforms and has a calculated molecular weight of approximately 61 kDa . The gene encoding human GPR123 maps to chromosome 10q26.3, while the mouse ortholog is located on chromosome 7 F4 .

Recent research has revealed that GPR123 plays an essential role in the maintenance and acquisition of pluripotency in human pluripotent stem cells (hPSCs) . Expression studies show GPR123 is predominantly found in:

• Adult and fetal brain

• Adult spinal cord

• Human testis tissue

What are the common applications for GPR123 antibodies in research?

GPR123 antibodies are valuable tools for multiple research applications, as summarized below:

Most commercially available GPR123 antibodies demonstrate reactivity with human samples, with many also cross-reacting with mouse and rat tissues . For optimal results, it is recommended that researchers titrate antibody concentrations for their specific experimental system .

What is the cellular and subcellular localization pattern of GPR123?

GPR123 displays distinct localization patterns that provide insight into its function:

• Cell surface localization: GPR123 is expressed at high levels at the surface of human embryonic stem cells (hESCs)

• Cytoplasmic expression: Residual staining is observed in the cytoplasm of hESCs

• Nuclear localization: Transmission electron microscopy (TEM) has revealed accumulation of GPR123 at the nucleus, nucleolus, and nuclear membrane of hESCs

• Tissue-specific expression: In human testis, immunohistochemical staining shows distinct positivity in spermatids

These localization patterns suggest multiple functional roles for GPR123 depending on cellular context and developmental stage.

How should GPR123 antibodies be stored and handled for optimal performance?

Proper storage and handling of GPR123 antibodies is critical for maintaining their performance:

Storage conditions:

• Store at -20°C for long-term preservation (stable for one year after shipment)

• For frequent use and short-term storage (up to one month), store at 4°C

• Avoid repeated freeze-thaw cycles which can compromise antibody integrity

Buffer composition:
Most commercial GPR123 antibodies are supplied in:

• PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• Some products contain 0.1% BSA in smaller sizes (20μl)

Important handling notes:

• Aliquoting upon receipt is recommended to minimize freeze-thaw cycles

• Some products contain sodium azide, which should be handled by trained staff only as it is hazardous

• For dilution, use fresh buffer solutions at appropriate pH

What optimization strategies improve GPR123 antibody performance in immunohistochemistry?

For optimal immunohistochemical detection of GPR123, consider the following protocol refinements:

Antigen retrieval methods:

• Primary recommendation: TE buffer pH 9.0

• Alternative approach: Citrate buffer pH 6.0

Dilution optimization:

• Begin with manufacturer's recommended range (typically 1:20-1:200 for IHC)

• Perform titration experiments to determine optimal concentration for your specific tissue

• Consider sample-dependent variables (fixation time, tissue type, expression level)

Visualization optimization:
For human testis tissue, GPR123 antibodies show distinct positivity in spermatids at dilutions of 1:50-1:200 . When staining other tissues, particularly neuronal samples, background reduction strategies may be necessary due to GPR123's high expression in neural tissues.

How can researchers validate the specificity of GPR123 antibodies?

Validating antibody specificity is crucial for obtaining reliable results. For GPR123 antibodies, consider these validation approaches:

Positive controls:

• Human testis tissue (shows distinct positivity in spermatids)

• hESCs (high expression at cell surface)

Negative controls:

• Embryoid bodies after 10+ days of differentiation (GPR123 expression decreases)

• Omit primary antibody but maintain all other steps

• Pre-absorption with immunizing peptide where available

Molecular validation:

• Correlation of IHC results with Western blot profiles showing expected MW (~61 kDa)

• RNAi knockdown of GPR123 should correlate with reduced antibody signal

• Comparison with mRNA expression data (e.g., qPCR)

How does GPR123 contribute to pluripotency maintenance in stem cells?

GPR123 plays a critical role in pluripotency maintenance, as evidenced by multiple experimental approaches:

RNAi studies in hPSCs:
When GPR123 is suppressed via RNAi in human pluripotent stem cells, the following consequences occur:

• Loss of pluripotency and initiation of differentiation

• Significant changes in colony morphology (increased cell size, dome-shaped formations)

• Accumulation of cells at the G2 phase of the cell cycle

• Absence of scratch closure in wound assay (decreased cell motility)

• Weak or absent staining for alkaline phosphatase (AP), a pluripotency marker

Temporal expression pattern:

• High GPR123 expression correlates with pluripotent status

• Expression begins to decrease from day 10 of embryonic body (EB) differentiation

• This temporal expression pattern further supports GPR123's role in maintaining pluripotency

What experimental approaches are effective for studying GPR123 function in reprogramming?

To investigate GPR123's role during cellular reprogramming, researchers have employed several sophisticated strategies:

RNAi during reprogramming initiation:

• Application of GPR123 RNAi during days 8-10 of the reprogramming process

• Results in decreased percentage of "true" hiPSC colonies (TRA-1-60+/CD44- population)

• By day 18, only 22.8% of cells were TRA-1-60+/CD44- compared to 86.6% in control groups

• Leads to reduced E-cadherin expression and decreased percentage of NANOG+ nuclei

• Prevents actin cytoskeleton remodeling required for successful reprogramming

Gene expression analysis:

• Comparison of GPR123 expression between different cell populations during reprogramming

• Analysis of TRA1-60+/CD44- sorted cells (representing "true" hiPSCs)

• Examination of TRA1-60+/CD44+ cells (representing intermediate reprogramming state)

These approaches demonstrate that GPR123 is not only important for maintaining pluripotency in established stem cell lines but is also critical during the acquisition of pluripotency during reprogramming.

What are the current analytical techniques for detecting GPR123 protein-protein interactions?

Understanding GPR123's interactions with other proteins is crucial for elucidating its function. Several techniques have been employed:

Co-localization studies:

• GPR123-Gαi co-localization has been observed during reprogramming

• Loss of this co-localization under GPR123 RNAi conditions suggests functional interaction

Immunoprecipitation approaches:

• While not explicitly detailed in the search results, standard co-immunoprecipitation (co-IP) using GPR123 antibodies would be appropriate for identifying protein binding partners

• For optimal results, use lysis buffers that preserve membrane protein interactions (containing mild detergents)

Proximity ligation assays:

• These could be employed to detect and visualize endogenous protein interactions in situ

• Particularly valuable for GPR123 given its membrane localization and potential transient interactions

How can researchers design effective GPR123 knockdown experiments?

Based on published research, effective GPR123 knockdown studies should consider:

RNAi approach optimization:

• Small interfering RNA (siRNA) has been successfully used to suppress GPR123 expression

• Normalize expression data using control genes such as GAPDH or RPL13A

• Verify knockdown efficiency at both mRNA level (qPCR) and protein level (Western blot)

Functional readouts:
Several established assays can measure functional consequences of GPR123 knockdown:

• Colony morphology assessment (dome-shaped formations indicate differentiation)

• Alkaline phosphatase staining (reduction indicates loss of pluripotency)

• Cell cycle analysis (accumulation at G2 phase)

• Wound scratch assay (impaired closure indicates decreased motility)

Temporal considerations:

• Changes in colony morphology may be observed as early as 2 days after GPR123 knockdown

• More pronounced effects become evident by day 4 (dome-shaped colonies, weak AP staining)

• For reprogramming studies, apply knockdown during days 8-10 and assess outcomes at day 18

What are common challenges when using GPR123 antibodies in Western blot applications?

When using GPR123 antibodies for Western blot analysis, researchers may encounter several challenges:

Protein extraction considerations:

• As a multi-pass membrane protein, GPR123 requires appropriate extraction methods

• Use lysis buffers containing mild detergents (e.g., 1% Triton X-100 or CHAPS)

• Complete solubilization is crucial to prevent aggregate formation

Expected band patterns:

• The calculated molecular weight of GPR123 is approximately 61 kDa

• Alternative splicing may result in additional bands

• Post-translational modifications might cause shifts in apparent molecular weight

Optimization strategies:

• For inconsistent results, adjust antibody dilution (recommended range: 1:500-1:1000)

• When detecting endogenous GPR123, increase protein loading (25-50 μg total protein)

• Consider using reducing agents in sample buffer to improve epitope accessibility

• For weak signals, extend exposure time or use enhanced chemiluminescence substrates

What control samples are recommended when studying GPR123 in different experimental contexts?

Selection of appropriate controls is crucial for accurate interpretation of GPR123 studies:

Positive control tissues/cells:

• Human testis tissue - shows distinct positivity in spermatids

• Human embryonic stem cells - high expression at cell surface

• Cell lines: HeLa, Raw264.7, and PC12 whole cell lysates have been used successfully in Western blot applications

Negative controls:

• Differentiated embryoid bodies (after day 10) show decreased GPR123 expression

• GPR123 knockdown samples serve as excellent negative controls

• Secondary antibody-only controls to assess non-specific binding

Expression benchmarks:

• GAPDH or RPL13A can be used as loading controls and for normalization in Western blot and qPCR experiments

• For immunohistochemistry, include adjacent sections with known GPR123 expression patterns

How can cross-reactivity issues with GPR123 antibodies be addressed?

Cross-reactivity can compromise experimental results. For GPR123 antibodies, consider these strategies:

Specificity assessment:

• Verify reactivity against intended species (human, mouse, rat)

• Some antibodies show reactivity with multiple species (human, mouse, rat) while others are human-specific

• Sequence alignment analysis can predict potential cross-reactivity with related G-protein coupled receptors

Blocking strategies:

• Use protein-free blocking reagents to reduce non-specific binding

• If cross-reactivity persists, pre-adsorption with the immunizing peptide (where available) can improve specificity

• Sequential antibody incubation protocols may help resolve signals from cross-reactive epitopes

Alternative detection methods:

• If a particular application shows persistent cross-reactivity, consider switching to another detection method

• Combining multiple detection methods provides stronger evidence of specificity

How might GPR123 antibodies contribute to understanding neurological disorders?

Given GPR123's expression in adult and fetal brain and spinal cord, antibodies against this receptor could be valuable tools for neurological research:

Potential research applications:

• Immunohistochemical mapping of GPR123 distribution in normal versus diseased neural tissues

• Investigation of GPR123's role in neurodevelopmental processes

• Exploration of changes in GPR123 expression in neurodegenerative disorders

Methodological considerations:

• Brain tissue often requires specialized fixation and antigen retrieval protocols

• Careful titration of antibody concentration is essential to balance specific signal and background

• Double immunofluorescence with neuronal/glial markers would help identify specific cell populations expressing GPR123

What is the potential for using GPR123 as a marker in stem cell characterization?

The essential role of GPR123 in pluripotency maintenance suggests its potential as a stem cell marker:

Marker validation studies:

• Correlation of GPR123 expression with established pluripotency markers (OCT4, NANOG, SOX2)

• Assessment of GPR123 expression during various differentiation protocols

• Evaluation of GPR123 as a predictive marker for reprogramming efficiency

Combined marker approaches:

• Integration of GPR123 into existing stem cell characterization panels

• Flow cytometry applications using fluorophore-conjugated GPR123 antibodies

• Multi-parameter analysis correlating GPR123 with functional pluripotency assays

What are the technical considerations for developing neutralizing antibodies against GPR123?

While current commercial antibodies are primarily detection reagents, developing neutralizing antibodies against GPR123 could provide valuable functional insights:

Target epitope selection:

• Focus on extracellular domains of GPR123 that might interact with ligands or neighboring proteins

• Structural analysis to identify accessible regions of the protein

• Custom peptide design representing specific functional domains

Validation strategies:

• Functional assays showing inhibition of pluripotency maintenance

• Competition assays with known GPR123 interactions

• Dose-response studies to determine potency and specificity

Application considerations:

• Cell permeability issues for targeting intracellular domains

• Need for careful negative controls (isotype-matched non-specific antibodies)

• Potential for combining with genetic approaches for comprehensive functional analysis

What resources are available for researchers studying GPR123?

Researchers interested in GPR123 can access various resources:

Antibody suppliers:

• Multiple vendors offer GPR123 antibodies with various applications and validations

• Pricing ranges from approximately €406 to $530 depending on quantity and supplier

Sequence resources:

• UniProt ID: Q86SQ6 (human GPR123)

• GenBank Accession Number: NM_001083909

• Gene ID (NCBI): 84435

Research model systems:

• Human embryonic stem cells (hESCs) - high GPR123 expression

• Human testis tissue - distinct positivity in spermatids

• Cell lines: HeLa, Raw264.7, and PC12 for Western blot applications

What protocols for GPR123 detection have been optimized and published?

Several optimized protocols for GPR123 detection are available:

Immunohistochemistry protocols:

• Antigen retrieval recommendation: TE buffer pH 9.0 or citrate buffer pH 6.0

• Dilution range: 1:20-1:200 for human testis tissue

Western blot protocols:

• Recommended dilution: 1:500-1:1000

• Use GAPDH as a loading control after membrane stripping

• Denature samples in standard SDS-PAGE conditions

Immunofluorescence:

• Dilution range: 1:200-1:1000

• Visualization of both membrane and nuclear localization has been achieved