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MagSi particles coated with lectins for glycoproteomics studies

Schematic of magnetic silica bead with a variety of functional conjugates.Schematic of magnetic silica bead with a variety of functional conjugates.
Figure 1. Schematic of magnetic silica bead with a variety of functional conjugates.

Alterations in protein glycosylation occur during development, and in the progression of many diseases including cancer, inflammation and autoimmune diseases. The recent field of glycoproteomics has emerged as an important tool in glycobiomarker discovery. The most frequently studied lectin for capture of glycosylated proteins is Concanavalin A (Con A). In addition, there exist ~20 more lectins with different individual selectivity towards glycosylated structures. Because of the variety of carbohydrates that are recognized by the lectin family, a comprehensive glycoproteomics study should include the ability to analyze multiple lectins.

Here we present a pilot study using MagDetect™ Magnetic Beads, Amine-NH2, 1um, (M2106-51) as a generic particle for the covalent attachment of any lectin. In this case wheat germ agglutinin (WGA) was used as the illustrative lectin (Figure 1). Covalent attachment of WGA to aminated MagDetect™ Magnetic Beads, Amine-NH2, 1um, (M2106-51) was carried out. For comparison, tosylated Dynabeads (Dynabeads M280, Tosylactivated) from another vendor were compared. (Tosylated Dynbeads were chosen for coupling in this study because the manufacturer reports a higher binding for the tosylated bead than for the amine-coupled magnetic bead.) The coupling itself has been performed according to the manufacturers protocols. Equal amounts of serum (50ug serum proteins) were applied to 5ul of WGA-coupled MagDetect™ Amine-NH2 beads and tosylated Dynabeads, followed by 30 minute incubation at 4°C.


SDS-PAGE of WGA-coupled magnetic beads using both MagDetect™ Amine-NH2 Magnetic Beads and tosylated Dynabeads.

Figure 2. SDS-PAGE of WGA-coupled magnetic beads using both MagDetect™ Amine-NH2 Magnetic Beads and tosylated Dynabeads.
Lane 1: Molecular Weight standards
Lanes 2,3: MagDetect™ Amine-NH2 Magnetic Beads coated with lectin WGA, boiled in SDS-PAGE buffer to release WGA. Single band is observed at ~18kD corresponding to WGA.
Lanes 4,5: Tosylated Dynabeads coated with WGA, treated with serum and boiled in SDS PAGE buffer to release all glycosylated proteins.
Lanes 6,7: MagDetect™ Amine-NH2 Magnetic Beads coated with WGA, treated with serum and boiled in SDS PAGE buffer to release all glycosylated proteins. Note ~5X greater binding (and release) of glycosylated proteins to MagDetect beads.
Lanes 8,9: Controls; non-glycosylated proteins adsorbed to both types of magnetic beads and treated as in lanes 4-7. No non-specific absorption observed

 

The binding efficiency of serum glycoproteins has been analyzed by an SDS-PAGE study (Fig. 2) and the results can be summarized as follows:
1) The WGA lectin was coupled successfully in high yield to the WGA-coupled MagDetect™ Amine-NH2 and tosylated Dynabeads (lanes 2,3).
2) No nonspecific binding of non-glycosylated serum proteins could be detected in the controls (lanes: 8,9).
3) The total amount of glycosylated proteins pulled out with the WGA-coupled MagDetect beads was ~5 times higher compared to the tosylated Dynabeads.
4) The coating and blocking of the tosylated Dynabeads takes ~40 hrs. whereas coating and blocking with MagDetect™ Amine-NH2 coupling chemistry, the process only takes ~1.5 h.
5) Using MagDetect™ Magnetic Beads, Amine-NH2, 1um, (M2106-51), it could be demonstrated that other lectins, such as ConA and JAC (Jacalin from Artocarpus integrifolia), could be coupled sufficiently as well (results not shown).

 

Conclusions
MagDetect™ Amine-NH2 Magnetic Beads provide an effective tool for any kind of lectin based glycoproteomics study. Efficient coupling of three widely used lectins (WGA, ConA and JAC) strongly indicates that the MagDetect™ Amine-NH2 particles are a superior tool for glycoproteomics studies.

 

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