159
Refold the purified denatured Clampbodies by stepwise dialysis
procedure as below.
1. Add 1.05 μL of 2-mercaptoethanol and 3 μL of 0.5 M EDTA
to 1.5 mL of purified solubilized protein.
2. Rotate gently at room temperature for 30 min to reduce disulfide bonds.
3. Inject protein solution into Slide-A-Lyzer Dialysis Cassettes,
3.5 K MWCO, 3 mL.
4. Dialyze protein solution by putting the Slide-A-Lyzer Dialysis
Cassettes into 1 L of Dialysis buffer containing 3 M Gdn-HCl.
Incubate at 4 °C for overnight (see Note 5).
5. Dialyze protein against Dialysis buffer containing 2 M GdnHCl at 4 °C overnight.
6. Dialyze protein against Dialysis buffer containing 1 M Gdn- HCl
at 4 °C overnight.
7. Dialyze protein against Dialysis buffer containing 0.5 M
Gdn- HCl at 4 °C overnight.
8. Dialyze protein against Dialysis buffer without Gdn-HCl at 4 °C
overnight.
9. Collect the contents in the cassette and centrifuge at 20,000 × g
for 5 min at 4 °C.
10. Collect the supernatant (see Note 6). Measure the concentration
of purified protein with a Pierce™ BCA protein assay kit as per
manufacturer’s instructions.
After the preparation of Clampbodies, perform SDS-PAGE and
Western blotting to confirm the purity and the amount of the
proteins (Fig. 1c, d).
1. Analyze the proteins by SDS-PAGE using a 5–20% gradient
gel and SDS running buffer.
2. Transfer the protein from the gel to a PVDF membrane with
Transfer buffer.
3. Incubate the membrane in TBST-S at 4 °C overnight.
4. Wash membrane three times with TBST.
5. Incubate the membrane with HRP-conjugated anti-His 6 antibody 4000-times diluted in TBST at 4 °C for 1 h.
6. Wash membrane three times with TBST.
7. Soak membrane in Amersham ECL Prime as per manufacturer’s instructions.
8. Detect the luminescence using a LAS-4000 mini
Luminoimager.
3.8 Refolding
of the Solubilized
Proteins
3.9 Western Blotting
Regulation of β-Lactamase Activity by Clampbody
Refold the purified denatured Clampbodies by stepwise dialysis
procedure as below.
1. Add 1.05 μL of 2-mercaptoethanol and 3 μL of 0.5 M EDTA
to 1.5 mL of purified solubilized protein.
2. Rotate gently at room temperature for 30 min to reduce disulfide bonds.
3. Inject protein solution into Slide-A-Lyzer Dialysis Cassettes,
3.5 K MWCO, 3 mL.
4. Dialyze protein solution by putting the Slide-A-Lyzer Dialysis
Cassettes into 1 L of Dialysis buffer containing 3 M Gdn-HCl.
Incubate at 4 °C for overnight (see Note 5).
5. Dialyze protein against Dialysis buffer containing 2 M GdnHCl at 4 °C overnight.
6. Dialyze protein against Dialysis buffer containing 1 M Gdn- HCl
at 4 °C overnight.
7. Dialyze protein against Dialysis buffer containing 0.5 M
Gdn- HCl at 4 °C overnight.
8. Dialyze protein against Dialysis buffer without Gdn-HCl at 4 °C
overnight.
9. Collect the contents in the cassette and centrifuge at 20,000 × g
for 5 min at 4 °C.
10. Collect the supernatant (see Note 6). Measure the concentration
of purified protein with a Pierce™ BCA protein assay kit as per
manufacturer’s instructions.
After the preparation of Clampbodies, perform SDS-PAGE and
Western blotting to confirm the purity and the amount of the
proteins (Fig. 1c, d).
1. Analyze the proteins by SDS-PAGE using a 5–20% gradient
gel and SDS running buffer.
2. Transfer the protein from the gel to a PVDF membrane with
Transfer buffer.
3. Incubate the membrane in TBST-S at 4 °C overnight.
4. Wash membrane three times with TBST.
5. Incubate the membrane with HRP-conjugated anti-His 6 antibody 4000-times diluted in TBST at 4 °C for 1 h.
6. Wash membrane three times with TBST.
7. Soak membrane in Amersham ECL Prime as per manufacturer’s instructions.
8. Detect the luminescence using a LAS-4000 mini
Luminoimager.
3.8 Refolding
of the Solubilized
Proteins
3.9 Western Blotting
Regulation of β-Lactamase Activity by Clampbody