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Serial lectin affinity chromatography
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Serial lectin affinity chromatography

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Category
Isolation & structural analysis of glycans
Protocol Name

Serial lectin affinity chromatography

Authors
Ohkura, Takashi
Department of Reproductive Biology, National Institute for Child Health and Development
KeyWords
Reagents

Lectin-immobilized gels are commercially available from Seikagaku Biobusiness Corp. (Tokyo, Japan), Sigma-Aldrich (St. Louis, MO), Vector Laboratories, Inc. (Burlingame, CA), and EY Laboratories (San Mateo, CA).

10 mM Tris-HCl buffer (pH 7.4), 0.02% sodium azide (TB)

TB containing an appropriate haptenic sugar, such as Con A; 10 mM or 0.2 M a-methylmannoside, RCA; 10 mM lactose, DSA; 1% N-acetylglucosamine oligomer, AAL; 5 mM fucose, UEA-I; 50 mM fucose, WFA; 10 mM N-acetylgalactosamine, PVL; 0.3 M N-acetylglucosamine, GS-I; 0.1 M galactose, TJA-I; 0.1 M lactose, MAL; 0.3 M lactose.

Ion-exchange resins AG-50W-X8 (H+, 50–100 mesh) and AG-4 (OH-, 100–200 mesh) (Bio-Rad Laboratories, Hercules, CA)

Instruments

Disposable 2 mL polystyrene column (Pierce, Rockford, IL)

Centrifugal vacuum concentrator

Liquid scintillation counter: PerkinElmer MicroBeta TriLux 1450 (PerkinElmer, Waltham, MA) or ALOKA LSC-6000 (Tokyo, Japan)

Methods
1.

Serial lectin affinity chromatography

1) 

 Wash the lectin column with 5 column volumes of TB supplemented with the respective haptenic sugar.

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2) 

 Equilibrate the column with 20–50 column volumes of TB.

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3) 

 Set the lectin column on a 10 mL tube.

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4) 

 Dissolve 3H-labeled sample in 200 μL of TB and apply to the column.

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5) 

 Stand for 10 min, add 800 μL of TB to the column and collect the eluent in the tube.

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6) 

 Move the column to the next tube and add 1mL of TB.

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7) 

 Repeat step 6) 3–8 times.

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8) 

 Move the column to the next tube, add 1mL of TB containing the respective haptenic sugar, and collect the eluent in the tube.

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9) 

 Stand for 10 min and then repeat step 8) 4 times.

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10) 

 Take aliquots from each fraction in counting vials, add and mix scintillation cocktail and measure radioactivity using a liquid scintillation counter.

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11) 

 Collect the flow-through fraction(−), the retarded fraction(r), and the bound fraction(+), and dry each separately by using a centrifugal vacuum concentrator.

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12) 

 If high-concentrated salts interfere with the following analysis, desalt the pooled fraction with a disposable column packed with AG-50W(H+) and AG-4(OH-) resins.

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Notes
  • Refer to the literature detailing the carbohydrate binding specificities of lectins.
  • To remove the haptenic sugar from the fraction, {make} prepare a mixture of sample:ethanol:butanol with a ratio of 1:1:4, and then apply the mixture to a Sepharose CL-4B column (bed volume, 1 mL, GE Health Care) pre-equilibrated with water:ethanol:butanol (1:1:4). After washing the column with 5 mL of water:ethanol:butanol (1:1:4), the oligosaccharide is eluted with 3 mL of 50% ethanol.
  • The order of the lectin columns used for the serial lectin chromatography is not very important; the author often uses them in the following order: WFA→ConA→RCA→DSA→AAL.
  • Jacalin and PNA, both of which recognize the core portion of O-glycans, are not suitable for the separation of released oligosaccharides. However, several other lectins are available for the analysis of the side chain of O-glycans.
  • The binding capacities of the lectins for oligosaccharides is almost nmole/mL gel depending on the lectin gel.
  • Glycoproteins generally bind more strongly to the lectin-immobilized gels than the released oligosaccharides. Moreover, the binding is stronger at lower temperature.
Figure & Legends

Figure & Legends

Fig. Procedure and result of serial lectin affinity chromatography

HuH-7 hepatoma cells were metabolically-labeled with [3H]glucosamine, the glycoproteins secreted in the culture medium were digested with N-glycanase to release the N-glycans. The N-glycans were digested with sialidase and then subjected to serial lectin chromatography. Although various types of glycans are released, mainly one component is purified using the combination of the serial lectin chromatography and Bio-gel P-4 gel-permeation chromatography. As a result, the structure of the sugar chain can be deduced from the carbohydrate binding specificities of the lectins and the sizes of the glycans.

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