2. After 16 h of growth, transfer 1 mL of the bacterial culture into
1 L of LB media containing ampicillin and incubate at 37
C
until an OD 600nm of 0.4–0.6 is reached (approximately 3 h).
3. Once an OD 600nm of 0.4–0.6 has been reached, induce gene
expression using 100 μM IPTG.
4. Transfer the culture to a 22
C shaker and incubate for an
additional 20 h.
5. Following the incubation, measure the OD 600nm of the culture
and harvest 500 mL of the cell culture via centrifugation for
20 min at 4000 rpm (Eppendorf 5415D centrifuge) and 4
C.
6. After removing the supernatant, resuspend the cell pellet in
20 mL of buffer A and place on ice.
7. Lyse the cells using sonication (Model CL-18, Fisher Scientific) at an amplitude of 40–50. Cells should be sonicated 10 s
on and 30 s off for 10 cycles.
8. Following sonication, centrifuge the cells for 20 min at
4000 rpm and 4
C and transfer the supernatant to microcentrifuge tubes.
9. Centrifuge the tubes at 13,000 rpm for 10 min to remove any
remaining debris and collect the supernatant into a single
fresh tube.
10. Purify the overexpressed protein(s) using a prepared HisTrap
HP column (GE Healthcare) using a syringe protocol (see
Note 4).
11. The fractions collected should then be evaluated for protein
content using a plate reader to measure absorbance at 280 nM.
Analyze fractions containing a spike in absorbance, typically
observed at the ~100 mM imidazole wash step, with SDS
PAGE using a 10% acrylamide gel.
12. After SDS PAGE is completed, stain the gel with Coomassie
Blue for 1 h then wash with deionized water to visualize
protein bands. Fractions containing the proteins of interest
can be identified using a protein ladder.
13. Combine fractions containing substantial quantities of the
proteins of interest with little contamination into a single
fresh tube.
14. Dialyze the combined protein fractions overnight into PBS at
4
C.
15. Measure the protein concentration within the solution using
the Bradford Assay with a clear polystyrene plate. Mix 5 μL of
the extract with 250 μL of the Bradford reagent and incubate at
room temperature for 20 min. Afterward, measure the absorbance at 595 nm and compare to a standard curve containing
0–1 mg/mL of BSA.
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Ruiquan Qi et al.
1 L of LB media containing ampicillin and incubate at 37
C
until an OD 600nm of 0.4–0.6 is reached (approximately 3 h).
3. Once an OD 600nm of 0.4–0.6 has been reached, induce gene
expression using 100 μM IPTG.
4. Transfer the culture to a 22
C shaker and incubate for an
additional 20 h.
5. Following the incubation, measure the OD 600nm of the culture
and harvest 500 mL of the cell culture via centrifugation for
20 min at 4000 rpm (Eppendorf 5415D centrifuge) and 4
C.
6. After removing the supernatant, resuspend the cell pellet in
20 mL of buffer A and place on ice.
7. Lyse the cells using sonication (Model CL-18, Fisher Scientific) at an amplitude of 40–50. Cells should be sonicated 10 s
on and 30 s off for 10 cycles.
8. Following sonication, centrifuge the cells for 20 min at
4000 rpm and 4
C and transfer the supernatant to microcentrifuge tubes.
9. Centrifuge the tubes at 13,000 rpm for 10 min to remove any
remaining debris and collect the supernatant into a single
fresh tube.
10. Purify the overexpressed protein(s) using a prepared HisTrap
HP column (GE Healthcare) using a syringe protocol (see
Note 4).
11. The fractions collected should then be evaluated for protein
content using a plate reader to measure absorbance at 280 nM.
Analyze fractions containing a spike in absorbance, typically
observed at the ~100 mM imidazole wash step, with SDS
PAGE using a 10% acrylamide gel.
12. After SDS PAGE is completed, stain the gel with Coomassie
Blue for 1 h then wash with deionized water to visualize
protein bands. Fractions containing the proteins of interest
can be identified using a protein ladder.
13. Combine fractions containing substantial quantities of the
proteins of interest with little contamination into a single
fresh tube.
14. Dialyze the combined protein fractions overnight into PBS at
4
C.
15. Measure the protein concentration within the solution using
the Bradford Assay with a clear polystyrene plate. Mix 5 μL of
the extract with 250 μL of the Bradford reagent and incubate at
room temperature for 20 min. Afterward, measure the absorbance at 595 nm and compare to a standard curve containing
0–1 mg/mL of BSA.
468
Ruiquan Qi et al.