IGSF21 Antibody

What is the molecular structure of IgSF21 and how does it interact with binding partners?

IgSF21 contains multiple immunoglobulin-like domains, with the Ig1 domain being both necessary and sufficient for neurexin2α binding. AlphaFold2 modeling has predicted the binding interface between IgSF21's Ig1 domain and neurexin2α's LNS1 domain. The interaction involves β-sheets (R53 to D76) of the IgSF21 Ig1 domain facing helical protrusions (L161 to Y171) found within the β11-β12 loop of the neurexin2α LNS1 domain . This interface is maintained primarily through hydrogen bonds, with IgSF21 utilizing side chains (R55, E56, Y60, K71, D76, H117) to interact with neurexin2α LNS1, alongside some hydrophobic interactions between Ig1 (V58, I68) and LNS1 (P98, L161) .

How has IGSF21 been identified as a binding partner for neurexin2α?

Multiple complementary approaches have confirmed the specific interaction between IgSF21 and neurexin2α:

• Unbiased pulldown assays coupled to tandem mass-spectrometry analysis using IgSF21-Fc protein as bait, with candidates selected based on a Bayesian false discovery rate (BFDR) ≤ 0.03

• Co-immunoprecipitation from mouse brain crude synaptosomal fractions showing anti-IgSF21 co-immunoprecipitated endogenous α-Nrxns

• Direct protein interaction assays using highly purified recombinant proteins showing Nrxn2α-Fc, but not Nrxn1β-Fc or Fc alone, pulled down IgSF21-His

• Surface plasmon resonance (SPR) demonstrating that soluble Nrxn2α bound immobilized IgSF21 with low nanomolar concentration, fitting a model of bimolecular (1:1) association

What is known about IGSF21 expression in neural tissues?

Studies examining IGSF21 expression, particularly in the context of diabetic retinopathy, have revealed changes in both mRNA and protein levels in retinal amacrine cells. Research has demonstrated statistically significant differences in IGSF21 expression between control and diabetic groups, as well as between diabetic groups at different time points (indicated by statistical markers *P<0.05 vs. control, #P<0.05 vs. group A, &P<0.05 vs. group B) . These findings suggest IGSF21 expression is dynamically regulated in neural tissues and may be altered under pathological conditions.

What are the most effective methods for detecting IGSF21 protein expression?

Several complementary techniques have been validated for IGSF21 detection:

• RT-PCR: Used with primers designed by Primer 5.0. For IgSF21, forward primer 5'-CTAAGCTCCAGCTACTGC-3' and reverse primer 5'-CATGACTGCATAACGCTGAC-3' have been successfully employed .

• Western blot: Protocols typically include:

  • Protein extraction using tissue lysate (80 mg tissue in 2 mL lysate)

  • Concentration measurement via BCA method

  • SDS-PAGE separation (12% gels, 25 μg protein per well)

  • Transfer to PVDF membrane

  • Blocking with TBST containing 5% skim milk powder

  • Primary antibody: goat anti-mouse IgSF21

• Immunofluorescence:

  • Cell permeabilization with 0.1% Triton (15 minutes)

  • Blocking with 10% goat serum (30 minutes)

  • Primary antibody: rabbit anti-mouse IgSF21 (1:500 dilution, 6-hour incubation)

  • Secondary antibody: FITC-labeled goat anti-mouse (1:500 dilution, 2-hour incubation)

  • Nuclear counterstaining with DAPI (8 minutes)

What protocols are recommended for studying IGSF21-neurexin2α interactions?

Research has employed several methods to investigate this interaction:

• Co-immunoprecipitation:

  • Culture amacrine cells to 60-80% confluence

  • Transfect with pCMV-Myc-IgSF21 and pCMV-HA-neurexin2α

  • Incubate for 24 hours at 37°C

  • Centrifuge at 1,500 rpm for 10 minutes

  • Lyse with pre-cooled RIPA solution (30 minutes on ice)

  • Add anti-IgSF21 or anti-neurexin2α antibodies (IgG as negative control)

  • Agitate for 6 hours, add protein A/G, agitate for 5 hours

  • Perform Western blot to analyze protein content

• Pulldown assays:

  • Use highly purified recombinant proteins (IgSF21-His with Nrxn2α-Fc)

  • Include appropriate negative controls (Nrxn1β-Fc, Fc alone)

• Surface plasmon resonance (SPR):

  • Immobilize purified IgSF21 on biosensor chip

  • Test binding to soluble Nrxn2α ectodomain (0-200 nM)

  • Analyze data fitting to bimolecular (1:1) association model

How can researchers generate and validate mutant constructs of IGSF21?

The following approaches have been documented:

• Site-directed mutagenesis:

  • Use IgSF21-HA and pDisplay-Nrxn2α LNS1-3 as templates

  • Design primers to introduce specific mutations, particularly targeting residues involved in the binding interface:

    • IgSF21 residues: R55, E56, Y60, K71, D76, H117 (hydrogen bonding); V58, I68 (hydrophobic interactions)

    • Neurexin2α residues: R160, L161, S162, S167, Y171; P98, L161

• Construct generation:

  • Extract RNA from amacrine cells

  • Design primers with restriction enzyme sites (BamH1, EcoR1)

  • Amplify target sequences by PCR

  • Digest products and vectors with restriction enzymes

  • Ligate products into appropriate vectors (pCMV-Myc, pCMV-HA)

• Validation methods:

  • Binding assays with purified proteins

  • Cell-based assays with HA-tagged constructs

  • Functional assays measuring effects on presynaptic differentiation

How does IGSF21 contribute to inhibitory synapse formation and function?

IGSF21 functions as a high-affinity receptor for neurexin2α, specifically promoting GABAergic presynaptic differentiation . The molecular mechanism involves:

• Direct protein-protein interaction between postsynaptic IGSF21 and presynaptic neurexin2α

• Binding specifically mediated through the Ig1 domain of IGSF21 and the LNS1 domain of neurexin2α

• Complex formation at nanomolar affinity, indicating physiologically relevant interaction

• Triggering of signaling cascades that organize GABAergic presynaptic terminals

This process is critical for establishing proper inhibitory circuit function in the nervous system. Research examining IGSF21 overexpression effects on miniature inhibitory postsynaptic currents (mIPSCs) in amacrine cells further supports its role in modulating inhibitory synaptic transmission .

What are the implications of IGSF21 dysfunction in neural disorders?

Given IGSF21's role in GABAergic synapse formation, dysfunction may contribute to disorders characterized by excitatory/inhibitory imbalance. Research has specifically examined IGSF21 in diabetic retinopathy (DR), revealing:

• Altered expression patterns in DR rat models following streptozotocin injection

• Changes in both mRNA and protein levels in retinal amacrine cells

• Statistical significance between control and diabetic groups at different time points

These findings suggest IGSF21 may play a role in the pathophysiology of diabetic retinopathy, potentially through disruption of inhibitory synaptic organization. Similar mechanisms could be relevant in other neurological conditions involving synaptic dysfunction.

What experimental models are optimal for studying IGSF21 function?

Several experimental systems have proven effective:

• In vivo models:

  • Sprague-Dawley rats injected with streptozotocin for diabetic retinopathy studies

  • Groups divided based on feeding periods to study disease progression

• Primary cell cultures:

  • GABAergic amacrine cells extracted from normal rats

  • Controlled cell density (5×10^5/mL)

  • Transfection with IGSF21 constructs to study overexpression effects

• Molecular tools:

  • Recombinant protein expression: IgSF21-HA, IgSF21-Fc, IgSF21-His

  • Neurexin constructs: pDisplay-Nrxn2α LNS1-3, Nrxn2α-Fc

  • Control constructs: HA-CD4, IgSF21-ires-GFP

These models provide complementary approaches to study IGSF21 from molecular interactions to cellular and systemic effects.

What are common challenges when using IGSF21 antibodies and how can they be addressed?

While the search results don't directly discuss antibody troubleshooting, standard considerations apply:

• Specificity concerns:

  • Validate using multiple antibodies targeting different epitopes

  • Include positive controls (tissues known to express IGSF21) and negative controls

  • Consider peptide competition assays to confirm specificity

• Application optimization:

  • For Western blot: Optimize primary antibody concentration (typically 1:500 has been effective)

  • For immunofluorescence: Include permeabilization step (0.1% Triton for 15 minutes) and adequate blocking (10% goat serum for 30 minutes)

  • For co-IP: Extended incubation times (6 hours for primary antibody, 5 hours for protein A/G)

• Isoform considerations:

  • Be aware of which isoform your antibody targets (IgSF21 L or S)

  • For binding studies, target regions outside the Ig1 domain to avoid interference with neurexin2α interaction

How can researchers quantitatively assess IGSF21-neurexin2α binding?

Surface plasmon resonance (SPR) has been effectively used to characterize this interaction :

• Experimental setup:

  • Immobilize purified IgSF21 on biosensor chip

  • Test binding to soluble Nrxn2α ectodomain at concentrations from 0-200 nM

  • Analyze sensorgrams fitted to 1:1 biomolecular interaction model

• Statistical analysis:

  • For comparing binding parameters between wild-type and mutant proteins:

    • Normal distributions: Unpaired Student's t-tests (equal variance) or Welch's t-tests (unequal variance)

    • Non-normal distributions: Mann-Whitney tests

    • Multiple comparisons: Welch's ANOVA with Dunnett's T3 post hoc analysis or Kruskal-Wallis tests with Dunn's post hoc analysis

• Data reporting:

  • Present as mean ± SEM from at least three independent experiments

  • Define statistical significance as p < 0.05

What controls should be included when studying IGSF21 in experimental settings?

Proper controls are essential for rigorous IGSF21 research:

• For antibody validation:

  • Rabbit IgG as negative control in immunoprecipitation experiments

  • Comparison of multiple antibodies targeting different epitopes

• For binding studies:

  • Fc alone and unrelated proteins (e.g., Nrxn1β-Fc) as negative controls in pulldown assays

  • Concentration series of binding partners (0-200 nM) for kinetic analyses

• For expression studies:

  • Control animals/tissues matched for age and other variables

  • Appropriate housekeeping genes (GAPDH) as internal controls for PCR

  • Multiple time points to capture dynamic expression changes

• For functional studies:

  • HA-CD4 as control for non-specific effects of overexpression

  • Multiple statistical tests appropriate to data distribution

What are promising research avenues for IGSF21 in neural system disorders?

Based on current knowledge, several research directions merit investigation:

• Expanded disease models:

  • Beyond diabetic retinopathy, examine IGSF21 expression and function in other neurological disorders involving inhibitory dysfunction

  • Develop conditional knockout models to assess region-specific functions

• Structural biology:

  • Experimental determination of IGSF21-neurexin2α complex structure to validate computational predictions

  • Structure-guided development of molecules that modulate this interaction

• Circuit-level analysis:

  • Investigate how IGSF21-mediated inhibitory synapse formation affects neural circuit development and function

  • Examine consequences of IGSF21 manipulation on behavioral phenotypes

How might advanced techniques enhance IGSF21 research?

Emerging methodologies could address current knowledge gaps:

• Single-cell analyses:

  • Single-cell RNA sequencing to identify cell populations expressing IGSF21

  • Spatial transcriptomics to map IGSF21 expression patterns in complex tissues

• High-resolution imaging:

  • Super-resolution microscopy to visualize IGSF21 localization at synapses

  • Live imaging to track IGSF21 dynamics during synapse formation

• Functional genomics:

  • CRISPR/Cas9-mediated editing to introduce specific mutations in the binding interface

  • Targeted manipulation of IGSF21 expression in specific neural populations

These approaches would provide mechanistic insights into IGSF21 function and potential therapeutic applications in disorders affecting inhibitory neurotransmission.