N -Glycoprotein Enrichment by Lectin Affinity Chromatography - Plant Proteomics: Methods and Protocols - page 608

Fig. 2 Different modalities of lectin affi nity chromatography. Lectin enrichment
can be done in parallel ( a ), sequentially ( b ), and using a mix of several lectins
( c ). The type of lectins used can be tailored to the kind of glycoprotein that is
being targeted

and as mixtures [ 27 ], as illustrated in Fig. 2 . If the main goal is to
enrich for N -glycoproteins an effective option is the use of mannose
binding lectins (Table 1 ).

Prepacked PGC columns (1 mL, brand name Hypersep Hypercarb)
can be purchased from Thermo Scientifi c.

2.2 Porous Graphitic
Carbon (PGC) Columns

2.3 Buffer Solutions

Buffer solutions must be prepared with bi-distilled water, fi ltered
with 0.45 m fi lters, degassed, and precooled to 4 °C before start-

ing the main protocol.

μ

1. Stock buffers: prepare 1 M Tris, pH 7.0 (Solution A) and 5 M
NaCl (Solution B) solutions. Dissolve 121.4 g Tris in 900 mL
water, and adjust the pH with HCl, and the fi nal volume to
1 L with water. Dissolve 292 g NaCl in 500 mL water and
adjust the volume to 1 L.
2. Protein Extraction buffer: 25 mM Tris, pH 7.0, 0.5 M NaCl,
0.2 M CaCl 2 , and 20 L/g fresh weight protease inhibitor

cocktails. For 100 mL of protein extraction buffer mix: 2.5 mL
A solution, 10 mL B solution, 2.94 g CaCl 2 , and 20

μ

L/g
fresh weight protease inhibitor cocktails and adjust the volume
to 100 mL ( see Note 1 ).
3. Binding buffer (Lectin): 20 mM Tris-HCl, 0.5 M NaCl, 1 mM
CaCl 2 , 1 mM MnCl 2 , and 1 mM MgCl 2 . Mix 2 mL Solution

μ