B Cell-based Small-angle Scattering Assay

Brief Summary

Small-angle X-ray scattering (SAXS) is a valuable structure analysis technique applied to acquire data on the shape, structure changes, and size of macromolecules in solution. SAXS is typically used to study the interactions between macromolecules and the functional mechanisms of molecules in cells. This method can offer low-resolution structural graphs of bound proteins, such as antigen-antibody complex, on the shape, conformation, and binding state.

Fig.1 Schematic representation of a SAXS experiment.²

Cell-based Small-angle Scattering Assay Service at Creative Biolabs

The powerful X-ray scattering techniques provided us an insight into the complex interaction between the specific antibody and peptides. A SAS model of the antibody and peptides complex could indicate the shape information on the spatial arrangement of antibodies on an antigen, antibody binding sites, and conformation.

Workflow

Key Features of the Small-angle Scattering Assay

Highlights

Our Capabilities

Data Analysis Software on Our Instrument

Function	Software	Description
Experimental data processing	PRIMUS, GNOM, CHROMIXS	Primary data processing
Ab initio modeling	MONSA, GASBOR, DAMMMIN, DAMMIF	Determine shape using dummy atom model
Rigid body modeling	CORAL, SASREF, BUNCH, GLOBSYMM	Model the multiple motif ensembled complexes
Structure refines	SREFLEX	Refine high-resolution protein models
Model evaluation and manipulation	CRYSOL, SRYSON, CIFSUP, DAMAVER, SASpy	Computationally model the scattering pattern

Case Study

Analysis of SARS-CoV-2 Novel Neutral Antibody Binding Site on Spike RBD

Background

Research Objectives

SAXS Assay Parameters
Instrument: EMBL P12 beamline of the storage ring PETRA III
Scatter detector: Pilatus-6M pixel
Protein solution concentration range: 0.3–4 mg/ml

Results

Fig.2 Applying SAXS to characterize the interaction between antibody-Sb23 and spike RBD.¹

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References

Custódio, T. F.; et al. Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2. Nature communications. 2020, 11(1): 5588.
Kikhney, A. G.; Svergun, D. I. A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS letters. 2015, 589(19 Pt A): 2570–2577.
Manalastas-Cantos, K. et al. ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis. J. Appl. Cryst. 2021, 54: 343-355.

For Research Use Only | Not For Clinical Use