Biology Reference
In-Depth Information
2.4 Affi nity
chromatography on
Concanavalin A
1. Concanavalin A (ConA) agarose conjugate (catalog number
C7555, Sigma-Aldrich) (
see
Note 4
).
2. ConA prewashing buffer B3: 20 mM Tris-HCl pH 7.4, 1 M
NaCl, 3.3 mM MgCl
2
/MnCl
2
/CaCl
2
. Mix 2 mL of 1 M
Tris-HCl pH 7.4, 20 mL of 5 M NaCl, 33 mL of 10 mM
MgCl
2
/MnCl
2
/CaCl
2
stock solutions. Add water to a volume
of 100 mL. Store at 4 °C.
3. ConA equilibration and washing buffer B1: 20 mM Tris-HCl
pH 7.4, 150 mM NaCl, 1 mM MgCl
2
/MnCl
2
/CaCl
2
. Mix
20 mL of 1 M Tris-HCl pH 7.4, 30 mL of 5 M NaCl, 100 mL
of 10 mM MgCl
2
/MnCl
2
/CaCl
2
stock solutions. Add water to
a volume of 1 L. Store at 4 °C.
4. ConA elution buffer B4: 20 mM Tris-HCl pH 7.4, 150 mM NaCl,
1 mM MgCl
2
/MnCl
2
/CaCl
2
, 1 M methyl-
-
D
-glucopyranose
(catalog number M9376, Sigma-Aldrich). Weigh 9.7 g of
methyl-
α
-
D
-glucopyranose. Add buffer B1 to a volume of
50 mL. Store at 4 °C.
5. Desalting columns: Econo-Pac 10 DG columns (catalog number
732-2010, Bio-Rad).
6. Desalting buffer B2: 0.1 M ammonium formate. Weigh 6.3 g
of ammonium formate and add water to a volume of 1 L.
Store at 4 °C.
7. Lyophilizator Christ Alpha 1-4 LSC (Fischer Scientifi c).
α
2.5 Separation
of Proteins by
SDS- PolyAcrylamide
Gel Electrophoresis
1. Mini-PROTEAN
®
Tetra Cell (Bio-Rad).
2. Resolving gel buffer stock solution: 1.5 M Tris-HCl pH 8.8.
Weigh 36.3 g of Tris (PlusOne™, catalog number 17-1321-
01, GE Healthcare). Add water to a volume of 180 mL. Mix
and adjust pH with HCl. Make up to 200 mL with water.
Store at 4 °C.
3. Stacking gel buffer stock solution: 1.25 M Tris-HCl pH 6.8.
Weigh 15 g of Tris (PlusOne™, catalog number 17-1321-01, GE
Healthcare). Add water to a volume of 90 mL. Mix and adjust
pH with HCl. Make up to 100 mL with water. Store at 4 °C.
4. PlusOne™ acrylamide 40 % solution (catalog number 17-1303-01,
GE Healthcare).
5. PlusOne™
N,N
-methylenebisacrylamide 2 % solution (catalog
number 17-1306-01, GE Healthcare).
6. 10% SDS solution. Weigh 5 g of SDS (PlusOne™, catalog
number 17-1313-01, GE Healthcare). Add water to a volume
of 30 mL. Mix until total dissolution (
see
Note 5
). Complete
to a fi nal volume of 50 mL. Store at room temperature.
7. Ammonium persulfate (APS) 10 % solution. Weigh 1 g of APS
(PlusOne™, catalog number 17-1311-01, GE Healthcare).
Add water to a volume of 10 mL. Store 1 mL aliquots at −20 °C.
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