This article describes the application of Concanavalin A and it covalent coupling with magnetic beads.
Part 1. Concanavalin A
Concanavalin A-Coated Magnetic Beads (ConA beads), as the name suggests, are biomagnetic beads in which the plant lectin Concanavalin A (ConA) is coupled with superparamagnetic nanomaterials. The following is a brief introduction to ConA Magnetic Beads.
Concanavalin A (ConA), a plant lectin protein with no blood group specificity, was the first plant lectin protein to be isolated and purified and crystallized from cuttle bean (Canavalia ensiformis, Pennisetum maritimum) since 1936.
ConA has 2 main forms depending on the pH of the solution in which it is present: the α-2 homodimer or the α-4 homotetramer [2]. Under alkaline conditions (pH>7.0) it exists as a tetramer (consisting of four molecular weight 26 kDa subunits); under acidic conditions (pH 4.5-5.5) Con A dissociates into an activated dimeric structure (52 kDa). In addition, the function of ConA is affected by divalent cations, e.g., in the absence of metal ions (Ca2+ and Mn2+), its conformation and glycoprotein binding function cannot be realized[1].
Fig. (1). Molecular modeling of (A) ConA monomer, (B) ConA dimer, (C) ConA tetramer with mannose, glucose and metal ions.
Part 2. Beads
Biomagnetic beads are a class of magnetic microspheres with nanometer particle size, formed by polymers and inorganic magnetic nanoparticles. Magnetic beads can be classified into three main categories according to their structure: core-shell type structure, sandwich type structure, and diffuse type structure. Magnetic materials include pure iron powder, carbonyl iron, magnetic ore, orthoferrite, and iron cobalt alloy, et al.
Magnetic nanomaterials, because of their special effects, such as small size effect, surface effect and quantum size effect, etc., in the size of Fe3O4 nanoparticles is less than 30 nm, the interference of the thermal disturbance inside the nanoparticles is significant, and at this time, these nanoparticles show a special magnetic property, i.e., superparamagnetism. Superparamagnetic Fe3O4 nanoparticles are widely used in the biological industry due to their high quality properties, such as non-toxicity, good biocompatibility, unique magnetic targeting properties, and ease of heat generation in alternating magnetic fields.
In contrast, covalent coupling of ConA with microspheres for immobilization of biomolecules has the following advantages:
- High stability of covalent coupling binding for reproducibility;
- Ligand ConA binding on the surface of microbeads for target molecule interactions;
- Kinetic characterization of the solution environment, suitable for biological experimental manipulation；
Part 3. Application of ConA magnetic beads
As reported in the literature, the main applications of ConA magnetic beads are categorized into 3 scenarios, which are performing cytoplasmic membrane separation, glycoprotein enrichment, and separation of immobilized cellular aspects.
Application I: Cytoplasmic membrane separation
Using ConA magnetic beads affected by the activation of divalent cations, so that it exists in Ca2+ and Mg2+ solution environments, which possesses the function of affinity interaction of terminal α-D-mannosyl and α-D-glucosyl, used in the plasma membrane purification, is a simple and efficient way. For example, plasma membrane separation of cells or tissues is a key step to further obtain plasma membrane proteins. ConA is able to bind glycosylated proteins on cells, and this principle can be utilized to obtain higher purity plasma membrane. The main operation steps are: immobilization of ConA on magnetic beads by binding biotinylated ConA to streptavidin magnetic beads; incubation of cell membranes with ConA magnetic beads for 1h; adsorption on a magnetic rack; washing with TBS for 5 times; and elution of cytoplasmic membrane solution with eluent[3].
Fig 2. ConA magnetic beads purification steps for plasma membrane
Application II: Glycoprotein Enrichment
ConA is specific for mannose and glucose and recognizes α-conjugated mannose, which happens to be the “core oligosaccharide” of many serum and cell membrane glycoproteins. Therefore, it can be used in immunology to separate glycosylated molecules such as glycoproteins in cell or tissue lysates or serum.
A major procedure was that by cross-link aminosilanized magnetic nanopearls (MNPs) with ConA via a bifunctional linker, bis- N-hydroxysuccinimide linoleate (DSS), to obtain ConA magnetic beads; to terminate the non-specific binding of the magnetic nanopearls using methoxyethylene glycol (MEG), and to carry out the magnetic separation; to add the extract of the cellular membrane proteins digested with trypsin to incubate both of the ConA magnetic beads and the captured glycopeptides, and finally to elute the captured glycopeptides, and to carry out vacuum drying. ConA magnetic beads were both incubated, the ConA magnetic beads that had bound glycoproteins were collected by the magnetic rack, the non-glycopeptides were washed, and finally the captured glycopeptides were eluted and vacuum dried. This method allows in-depth analysis of specific glycosylation sites of tumor-associated proteins (e.g., EGFR)[4].
Fig 3. Superparamagnetic nanoparticles coupled to different lectins
Application III: Isolation of immobilized cells
The use of ConA magnetic beads (magnetic beads covalently bound to high-purity companion Concanavalin A) to bind to glycoproteins on cell membranes or nuclear membranes, thereby capturing cells or nuclei, allows visualization of experimental manipulation of small numbers of cells. For example, ConA magnetic beads are used in CUT&Tag and CUT&RUN [5] experiments, which are novel techniques used to study the structure and function of chromatin, and are immobilized by binding ConA magnetic beads to the cells to visualize the operation and avoid the problem of cell loss caused by centrifugation.
Compared to the traditional ChIP-seq technology for studying DNA-protein interactions, CUT&Tag and CUT&RUN have the following advantages:
- ConA magnetic beads bind to cell membrane glycoproteins to visualize the operation and enhance the experimental operation experience;
- No need for centrifugation, just ConA magnetic beads adsorbed on the magnetic rack to complete the separation of cell samples and solutions;
- Can be operated with as low as 10 cells, circumventing the need for a large number of samples for ChIP-seq.
Fig 4. Schematic diagram of ChIP-seq, CUT&Tag, CUT RUN experiment flow
Part 4. YEASEN Concanavalin A-Coated Magnetic Beads
ConA magnetic beads, developed by YEASEN, are capable of binding to glycoproteins, glycolipids, polysaccharides and other molecules with glycosylation modification in a fast, efficient, sensitive and specific manner after strict raw material selection and multiple process optimization and improvement. It is mainly used for cell isolation or for the isolation of glycosylated molecules such as glycoproteins in cell or tissue lysates or serum, and is especially directly used for experiments such as CUT &RUN and CUT&Tag (an innovative technology for ChIP-seq experiments).
1.Product Features
- Stable batch production and better reproducibility of results;
- Stable performance storage
- Cell capture efficiency>90%
2.Product Information
|
Cat NO. |
Cat#19810ES |
|
Size |
1mL/5mL/20mL |
|
Color |
Brownish yellow |
|
Bead concentration |
10 mg/mL |
|
Solid content |
9-11 mg/mL |
|
Bead size |
1 µm |
|
Capacity |
105 cells/µL beads |
3.Product Performance Data
(1)Monodispersity
Under the same treatment conditions and under the magnification of 10×/40×, the ConA beads were basically monodispersed, and no obvious agglomeration was observed relative to the competitor.
|
YEASEN Raw Material beads |
Competitor ConA beads |
YEASEN ConA beads（Cat#19810） |
Fig 5. Graph of monodispersity results
(2)Effect of ConA beads binding cell
The same number of cells were incubated with the beads for the same amount of time, and the number of cells remaining after conjugation of the ConA beads was automatically detected under the cell analyzer, and the results showed that the YEASEN ConA beads (Cat#19810) outperformed the competitor's products.
|
Cell suspension |
Cells remaining after binding of competitive ConA magnetic beads |
Cells remaining after binding of YEASEN ConA magnetic beads |
Fig 6. Picture of ConA magnetic bead bound cells
(3)Cell Binding Number and Repeatability
Both the 10µL YEASEN ConA Beads (Cat#19810) and the 10µL Competitor ConA Beads, bind E7 level numbers of cells comparable to the competitor. Cell capture was >90% under repeated operations.
Fig 7. The results of ConA beads binding cell number and capture rate
(4)Acceleration Stability
Dispense at 1 mL/tube. The storage conditions were: 4℃, 37℃ accelerated treatment for 2, 4, 7, 11 and 14 days, and -20℃ destruction treatment for 1, 3, 6 and 8 days. The results showed that under the same experimental conditions: the cell capture rate of the products stored at 4℃, accelerated treatment at 37℃ and destruction treatment at -20℃ was >95%, and the CV value of the three groups of replicates under each treatment condition was within 1%, which showed good reproducibility.
Fig 8. The results of Cell capture rate and repeatability bias of ConA beads under different temperature and time
Ordering information
|
19810ES |