Analytical Instruments
Thiol Coupling to mSAM or Dextran Surface
Chemicals:
PBST
Mix of 2M NaCl and 10 mM NaOH
EDC
NHS
Distilled, Deionized water
Cystamine HCl
0.1 M sodium phosphate pH 8.5
DTT or DTE
PDEA/NaCl
0.1 M sodium acetate pH 4.0
20 mM sodium acetate pH 5.2
Cysteine/NaCl
Procedure for Surface Thiol Coupling:
1.) Use PBST as running buffer and a flow rate of 0.05 mL/min @ 25C.
For longer injections and/or smaller loop sizes, adjust the flow rate down to 0.010 or
0.005 mL/min after an initial response is seen.
2.) Inject 2 M NaCl and 10 mM NaOH solution for 2-3 min over the chip to clean the
surface.
3.) Activate the surface by injecting a solution of 0.2 M EDC (40 mg/mL) and 0.05 M
NHS (10 mg/mL) in distilled, deionized H2O for 7 min over both the sample and
reference sides.
4.) Prepare a 40 mM solution of cystamine dihydrochloride (9 mg/mL) in 0.1 M sodium
phosphate pH 8.5. Inject over the sample side only for about 3 minutes.
5.) Prepare a 0.1 M solution of dithioerythritol (15.4 mg/mL) in 0.1 M sodium phosphate
pH 8.5. Inject over the sample side only for about 5 minutes.
6.) Inject your ligand at a concentration of 20-50 µg/mL in 20 mM sodium acetate pH
5.2 for about 30 minutes over the sample side only.
7.) Inject 20 mM PDEA (4.5 mg/mL) and 1 M NaCl in 0.1 M sodium acetate pH 4.0 for
about 8 minutes over both sample and reference sides.
Reichert, Inc., 3362 Walden Avenue, Depew, NY 14043
Telephone: 716 686 4500 • Fax: 716 686 4555 • Email: [email protected]
www. re ic h e rta i. c o m • I SO 9 0 0 1 R e g is te re d
–2–
February 26, 2013
Analytical Instruments
The procedure should look like this:
Im m o b iliz a tio n o f P ro te in a fte r D ia ly s is v ia T h io l C o u p lin g
3 6 4 uR I U o f P ro te in I m m o b iliz e d
19000
2 0 m M P D E A + 1 M N a C l in
0 .1 M N a A c e ta t e p H 4
17000
15000
0.2M E D C
0.1M NH S
(A c tiva tio n )
4 0 m M C y s t a m in e H C l
in 0 . 1 M N a P h o s p h a te
p H 8 .5
13000
0 .1 M D ith io e ry t h rit o l in 0 . 1 M N a
P hos phate pH 8.5
11000
9000
1 0 0 u g /m L D ia ly z e d P ro t e in
in 2 0 m M N a A c e t a te
7000
5000
3000
6000
7000
8000
9000
10000
11000
12000
13000
14000
Procedure for Ligand Thiol Coupling:
1.) Use PBST as running buffer and a flow rate of 0.05 mL/min @ 25C.
For longer injections and/or smaller loop sizes, adjust the flow rate down to 0.010 or
0.005 mL/min after an initial response is seen.
2.) Inject 2 M NaCl and 10 mM NaOH solution for 2-3 min over the chip to clean the
surface.
3.) Activate the surface by injecting a solution of 0.2 M EDC (40 mg/mL) and 0.05 M
NHS (10 mg/mL) in distilled, deionized H2O for 7 min over both the sample and
reference sides.
4.) Inject 80 mM PDEA (18 mg/mL) in 0.1 M sodium phosphate pH 8.5 for about 5
minutes over the sample side only.
5.) Inject your ligand at a concentration of 20-50 µg/mL in 20 mM sodium acetate pH
5.2 for about 30 minutes over the sample side only.
6.) Inject 50 mM cysteine (6.1 mg/mL) and 1 M NaCl in 0.1 M sodium acetate pH 4.0 for
about 5 minutes over both the sample and reference sides.
–3–
Analytical Instruments
The procedure should look as follows:
February 26, 2013