Isolation of mannan-binding protein from human serum (plasma) ~Method 2

Authors：

Category

Sugar binding proteins

Protocol Name

Isolation of mannan-binding protein from human serum (plasma) ~Method 2

Authors

Kawasaki, Nobuko
Research Center for Glycobiotechnology, Ritsumeikan University

KeyWords

Mannan-binding protein (MBP) Enzyme-linked lectin binding assay Lectin pathway Mannan-binding lectin (MBL) Mannan-agarose

Reagents

●	Pooled normal human serum
●	Mannan from S. cerevisiae (Sigma-Aldrich, St. Louis, MO)
●	CNBr-activated Sepharose 4B (GE-Healthcare, Little Chalfont, UK)
●	Sepharose 4B-mannan (yeast mannan-coupled with CNBr-activated Sepharose 4B)
●	2×Loading buffer for Sepharose 4B-mannan affinity column chromatography: 0.08 M imidazole-HCl buffer, pH 7.8, containing 2.5 M NaCl and 0.04 M CaCl₂*¹
●	Loading buffer for Sepharose 4B-mannan affinity column chromatography: 0.04 M imidazole-HCl buffer, pH 7.8, containing 1.25 M NaCl and 0.02 M CaCl₂*²
●	Elution buffer A for Sepharose 4B-mannan affinity column chromatography: 0.02 M imidazole-HC1 buffer, pH 7.8, containing 1.25 M NaCl and 2 mM EDTA *³
●	Elution buffer B for Sepharose 4B-mannan affinity column chromatography: 0.02 M imidazole-HC1 buffer, pH 7.8, containing 1.25 M NaCl, 0.02 M CaCl₂, and 0.05 M mannose *⁴
●	1 M CaCl₂

Instruments

●	Columns for open-column chromatography (column I, 2.6×30cm; column II, 1.0×20 cm; column III, 1.0×10 cm)
●	Ultrafiltration membrane filter (cut-off, 10 kDa)

Methods

Isolation of Mannan-binding Protein from human serum (plasma) ~Method 2

1)	Add an equal volume of 2× loading buffer for a mannan column (reagent *¹) to human serum (batch of 500 mL).	Comment 0

2)	Apply to a Sepharose 4B-mannan column (column I, gel volume: 100 mL), which has been washed with 3 column volumes of elution buffer A for a mannan column (reagent ³), and then equilibrate with one column volume of loading buffer for a mannan column (reagent ²).	Comment 1

3)	Wash the column with loading buffer (reagent *²) until the absorbance at 280 nm becomes less than 0.05 cm^-¹ (5 times the column volume) at a flow rate of 50 mL/h.	Comment 0

4)	Elute the protein bound to the column with approximately 4 column volumes of elution buffer A (reagent *³) at a flow rate of 15 mL/h.	Comment 0

5)	Add 1 M CaCl₂ to the eluate to a final concentration of 20 mM CaCl₂.	Comment 0

6)	Apply to a smaller second affinity column (column II, gel volume: 15 mL) of Sepharose 4B-mannan.	Comment 0

7)	Wash the column with loading buffer (reagent *²).	Comment 0

8)	Elute the protein bound to the column with elution buffer A (reagent *³).	Comment 0

9)	Add 1 M CaCl₂ to the eluate to a final concentration of 20 mM CaCl₂.	Comment 0

10)	Apply to a smaller third affinity column (column III, gel volume: 5 mL) of Sepharose 4B-mannan.	Comment 0

11)	Wash the column with loading buffer (reagent *²).	Comment 0

12)	Elute the bound proteins with 3 column volumes of elution buffer B (reagent ⁴), and then with 3 column volumes of elution buffer A (reagent ³).	Comment 1

13)

Subject an aliquot of the fractions eluted with elution buffer B (reagent *⁴) to SDS-PAGE (10% or 12.5% acrylamide gel) under reducing conditions, and pool the fractions comprising the single 31 kDa band as purified MBP.

Comment 0

Notes

Carry out all the procedures described below at 4°C.

Initial amount

1 L of human serum

Produced amount

> ~1 mg protein

Discussion

Through these procedures, approximately 1 mg of purified MBP is routinely obtained from 1 L of human serum with a recovery of approximately 30%, with 15,000–20,000 fold purification. Note the comments on the stability of the purified MBP for Method 1.

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Date of registration：2015-02-25 13:34:06