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Detection of binding of MGL to glycoproteins after separation by electrophoresis
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Detection of binding of MGL to glycoproteins after separation by electrophoresis

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Introduction Protocol References Credit lines
Category
Sugar binding proteins
Protocol Name

Detection of binding of MGL to glycoproteins after separation by electrophoresis

Authors
Fujihira, Haruhiko
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo

Usami, Katsuaki
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo

Irimura, Tatsuro *
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo
*To whom correspondence should be addressed.
KeyWords
Reagents

Samples (glycoproteins or lysates of cells)

All chemicals for gel electrophoresis in the presence of SDS

Transfer buffer for protein blotting

PVDF membrane (Merck Millipore, Billerica, MA)

The filter paper (Whatman 3MM chr, Whatman International Ltd., Kent, UK)

biotinylated recombinant human MGL (expressed in E. coli and purified on galactose-Sepharose according to Suzuki et al,1996 and biotinylated according to Kumamoto et al, 2004)

HRP-streptavidin (Invtrogen/Life Technologies, Carlsbad, CA)

blocking solution : 3 [w/v]% BSA in phosphate-buffered saline containing 0.01 [w/v]% sodium azide)

Washing buffer : 0.5% (w/v) Tween-20 in phosphate-buffered saline (PBST)

Methods
1.

SDS-PAGE

1) 

 Prepare glycoprotein samples.

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2) 

 SDS-PAGE on gels on appropriate density until the dye front is 0.5 cm from the bottom of the gel.

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2.

Protein blotting

1) 

 Before transfer, soak filter papers with transfer buffer and PVDF membranes with methanol.

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2) 

 Remove the gels carefully from glass plates.

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3) 

 Place a filter paper on the transfer apparatus, place a PVDF membrane on the filter paper, and place the gel on the membrane. Finally, place another filter paper on the gel. Make sure that there is no bubble left between them.

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4) 

 Start transfer under constant current conditions (2 mA/cm2 of membrane) for 90 min.

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3.

Binding of MGL

1) 

 After transfer of glycoproteins, remove membranes from the gels, place them into the blocking solution, and incubate them at room temperature for at least 30 min with gentle shaking.

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2) 

 Dissolve biotinylated-lectin (2.5 μg/mL) in 1 mL of PBST containing Ca2+ and Mg2+.

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3) 

 Wash the membranes with PBST at room temperature for 5 min.

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4) 

 Place the membranes into the solution of biotynilated-lectin and incubate at room temperature for 2 h or at 4ºC for over night.

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5) 

 Wash the membranes 6 times with PBST containing Ca2+ and Mg2+ for 5 min each.

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6) 

 Dilute 1 mg of HRP-sterptavidin into 1 mL of PBST containing Ca2+ and Mg2+.

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7) 

 Transfer the membranes into the HRP-strepeavidin solution and incubate at room temperature for 1–2 h.

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8) 

 Wash the membranes 6 times with PBST containing Ca2+ and Mg2+ for 5 min each.

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9) 

 Process for chemiluminescence detection of peroxidase activity according to the ECL method.

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Figure & Legends

Figure & Legends

Fig. 1. Lectin blotting of Virus-like particle (VLP) with Zaire Ebola viral glycoproteins (GPs).

VLP with Zaire Ebola viral GPs and Zaire Ebola viral GPs without mucin-like region were solubilized and the respective GP was separated by 10% SDS-PAGE and blotted with biotinylated-recombinant human MGL (right) and anti-GP antibody (ascites 42/3.7) (left). The amount of GP was quantified by the band intensity of anti-GP antibody blotting using ImageJ.

Z: Zaire Ebola viral GPs, Δ: Zaire Ebola viral GPs without mucin-like region

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