2 Materials
Prepare all buffers in ultrapure H 2 O, which is attained by filtering
deionized H 2 O through purification filters until it has a resistivity
reading of !18 MΩ cm at room temperature. All buffers and
proteins should be stored at 4
C when not in use. Follow all
institutional practices on waste disposal, particularly of the glass
capillaries used in the MST experiments. Use appropriate personal
protective gear (gloves, eyewear, etc.) throughout.
2.1 Buffers/
Detergent
1. 10Â phosphate-buffered saline (PBS): Place 80 g of NaCl, 2 g
of KCl, 17.8 g of Na 2 PO 4 · 2H 2 O, and 2.45 g of KH 2 PO 4 in a
1 L graduated cylinder, and fill to 950 mL. Adjust the pH to
7.4 using 12N HCl or 10 M NaOH as necessary. Bring the final
volume to 1.0 L with H 2 O. Pass through a 0.22-μm filter and
store.
2. 1Â PBS: We find that sometimes dilution from the 10Â stock
alters the final pH of the 1Â PBS. Therefore, we find it prudent
to check the pH upon dilution as follows. Add 100 mL of 10Â
PBS to 850 mL of H 2 O. Check the pH and adjust as necessary
using 12N HCl or 10 M NaOH. Bring the total volume to 1 L
with 0.22-μm filtered H 2 O.
3. 10% (v/v) Tween-20 (supplied by NanoTemper Technologies
GmbH when ordering capillaries, see below).
4. PBS–Tween: Mix 5 μL of 10% Tween-20 with 995 μL of 1Â
PBS (see Note 1).
2.2 Proteins
1. α-chymotrypsin (α-CT): Dissolve approximately 20 mg of the
protein with 1 mL of 1Â PBS. Place 500 μL into a 0.22-μm
centrifuge filter and centrifuge at a maximum of 5000 Â g for
5 min (see Note 2). Dilute the protein 100-fold (i.e., use a
dilution factor, f, of 100) and measure its absorbance at 280 nm
(A 280 ) using a spectrophotometer (see Notes 3–5). Using an
extinction coefficient (ε 280 ) of 51,840 M
À1 cm
À1 , calculate the
concentration of the stock using the following formula:
αÀCT ¼
A 280 Â f
ε 280 Â d
ð4Þ
where d is the path length of the cuvette used. Make
200 μL of 20 μM α-CT, 0.05% (v/v) Tween-20 by mixing
the proper volume of protein with 1 μL of 10% (v/v) Tween20 and the remaining volume of 1Â PBS.
2. Soybean trypsin inhibitor (SBTI): Dissolve approximately
20 mg of the protein with 1 mL of 1Â PBS. Follow the same
procedure as for α-CT to measure the concentration of the
164
Shih-Chia Tso and Chad A. Brautigam
Prepare all buffers in ultrapure H 2 O, which is attained by filtering
deionized H 2 O through purification filters until it has a resistivity
reading of !18 MΩ cm at room temperature. All buffers and
proteins should be stored at 4
C when not in use. Follow all
institutional practices on waste disposal, particularly of the glass
capillaries used in the MST experiments. Use appropriate personal
protective gear (gloves, eyewear, etc.) throughout.
2.1 Buffers/
Detergent
1. 10Â phosphate-buffered saline (PBS): Place 80 g of NaCl, 2 g
of KCl, 17.8 g of Na 2 PO 4 · 2H 2 O, and 2.45 g of KH 2 PO 4 in a
1 L graduated cylinder, and fill to 950 mL. Adjust the pH to
7.4 using 12N HCl or 10 M NaOH as necessary. Bring the final
volume to 1.0 L with H 2 O. Pass through a 0.22-μm filter and
store.
2. 1Â PBS: We find that sometimes dilution from the 10Â stock
alters the final pH of the 1Â PBS. Therefore, we find it prudent
to check the pH upon dilution as follows. Add 100 mL of 10Â
PBS to 850 mL of H 2 O. Check the pH and adjust as necessary
using 12N HCl or 10 M NaOH. Bring the total volume to 1 L
with 0.22-μm filtered H 2 O.
3. 10% (v/v) Tween-20 (supplied by NanoTemper Technologies
GmbH when ordering capillaries, see below).
4. PBS–Tween: Mix 5 μL of 10% Tween-20 with 995 μL of 1Â
PBS (see Note 1).
2.2 Proteins
1. α-chymotrypsin (α-CT): Dissolve approximately 20 mg of the
protein with 1 mL of 1Â PBS. Place 500 μL into a 0.22-μm
centrifuge filter and centrifuge at a maximum of 5000 Â g for
5 min (see Note 2). Dilute the protein 100-fold (i.e., use a
dilution factor, f, of 100) and measure its absorbance at 280 nm
(A 280 ) using a spectrophotometer (see Notes 3–5). Using an
extinction coefficient (ε 280 ) of 51,840 M
À1 cm
À1 , calculate the
concentration of the stock using the following formula:
αÀCT ¼
A 280 Â f
ε 280 Â d
ð4Þ
where d is the path length of the cuvette used. Make
200 μL of 20 μM α-CT, 0.05% (v/v) Tween-20 by mixing
the proper volume of protein with 1 μL of 10% (v/v) Tween20 and the remaining volume of 1Â PBS.
2. Soybean trypsin inhibitor (SBTI): Dissolve approximately
20 mg of the protein with 1 mL of 1Â PBS. Follow the same
procedure as for α-CT to measure the concentration of the
164
Shih-Chia Tso and Chad A. Brautigam