6. Sonicate the sample with an ultrasonicator (see Note 7). Centrifuge at 20,000 Â g for 10 min at 4
C and collect the
supernatant in a new tube. Keep the sample on ice.
7. Wash 2 mL of TALON
® resin (Clontech) with 10 mL of water.
Centrifuge at 700 Â g for 2 min at 4
C and then remove the
supernatant.
8. Equilibrate the resin with 10 mL of wash buffer. Centrifuge at
700 Â g for 2 min at 4
C and then remove the supernatant.
9. Apply the supernatant of the bacterial lysate to the equilibrated
resin. Incubate the mixture with gentle rotation for 20 min at
4
C. Centrifuge at 700 Â g for 5 min at 4
C and then remove
the supernatant.
10. Wash the resin with 10 mL of cold wash buffer three times (see
Note 8).
11. Elute the His-tagged protein with 12 mL of cold elution
buffer.
12. Check the yield and purity of the eluted proteins by
SDS-PAGE followed by Coomassie brilliant blue (CBB)
staining.
13. Dialyze the protein by 4 L of PBS for 2 h at 4
C and repeat
twice (see Note 9). Concentrate the protein until the concentration reaches 20 μM by centrifugal filter units. Sterilize
through a 0.22-μm filter. Store at À80
C.
3.2 Purification of
Recombinant Proteins
(GST-Tag)
1. Culture the BL21 (DE3) bacteria carrying the desired plasmid
(mouse MafA-11R-pGEX-6p-1) in 100 mL of LB medium
with 100 μg/mL ampicillin overnight at 37
C.
2. Decant the cultured bacteria into 900 mL of LB medium with
100 μg/mL ampicillin. Culture them again until the OD600
becomes approximately 0.8 at 37
C (see Note 4).
3. Add IPTG at a final concentration of 0.2 mM to induce the
expression of the desired protein. Continue the culture by
incubating at 32
C for 6–8 h (see Note 5).
4. Harvest the bacteria through centrifugation at 6000 Â g for
10 min at 4
C.
5. Add 50 mL of cold lysis buffer 1 to the bacterial pellets and
resuspend them by pipetting and vortexing.
6. Add 50 mL of cold lysis buffer 2 to the bacteria and then lyse
them with a vortex mixer. Incubate for 30 min on ice (see Note
6).
7. Sonicate the samples with an ultrasonicator (see Note 7). Centrifuge at 20,000 Â g for 10 min at 4
C and collect the
supernatant in a new tube. Keep the sample on ice.
90
Taku Kaitsuka and Kazuhito Tomizawa
C and collect the
supernatant in a new tube. Keep the sample on ice.
7. Wash 2 mL of TALON
® resin (Clontech) with 10 mL of water.
Centrifuge at 700 Â g for 2 min at 4
C and then remove the
supernatant.
8. Equilibrate the resin with 10 mL of wash buffer. Centrifuge at
700 Â g for 2 min at 4
C and then remove the supernatant.
9. Apply the supernatant of the bacterial lysate to the equilibrated
resin. Incubate the mixture with gentle rotation for 20 min at
4
C. Centrifuge at 700 Â g for 5 min at 4
C and then remove
the supernatant.
10. Wash the resin with 10 mL of cold wash buffer three times (see
Note 8).
11. Elute the His-tagged protein with 12 mL of cold elution
buffer.
12. Check the yield and purity of the eluted proteins by
SDS-PAGE followed by Coomassie brilliant blue (CBB)
staining.
13. Dialyze the protein by 4 L of PBS for 2 h at 4
C and repeat
twice (see Note 9). Concentrate the protein until the concentration reaches 20 μM by centrifugal filter units. Sterilize
through a 0.22-μm filter. Store at À80
C.
3.2 Purification of
Recombinant Proteins
(GST-Tag)
1. Culture the BL21 (DE3) bacteria carrying the desired plasmid
(mouse MafA-11R-pGEX-6p-1) in 100 mL of LB medium
with 100 μg/mL ampicillin overnight at 37
C.
2. Decant the cultured bacteria into 900 mL of LB medium with
100 μg/mL ampicillin. Culture them again until the OD600
becomes approximately 0.8 at 37
C (see Note 4).
3. Add IPTG at a final concentration of 0.2 mM to induce the
expression of the desired protein. Continue the culture by
incubating at 32
C for 6–8 h (see Note 5).
4. Harvest the bacteria through centrifugation at 6000 Â g for
10 min at 4
C.
5. Add 50 mL of cold lysis buffer 1 to the bacterial pellets and
resuspend them by pipetting and vortexing.
6. Add 50 mL of cold lysis buffer 2 to the bacteria and then lyse
them with a vortex mixer. Incubate for 30 min on ice (see Note
6).
7. Sonicate the samples with an ultrasonicator (see Note 7). Centrifuge at 20,000 Â g for 10 min at 4
C and collect the
supernatant in a new tube. Keep the sample on ice.
90
Taku Kaitsuka and Kazuhito Tomizawa