Determination of target protein solubility
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1112
Materials
LB medium
Kanamycin stock solution
Ampicillin stock solution
Lysis buffer for purification under native conditions
1´ PAGE sample buffer
2´ PAGE sample buffer
Culture growth
1. Inoculate 10 ml LB medium containing 100 mg/ml ampicillin and 25 mg/ml kanamycin in a 50-ml flask. Grow the cultures overnight at 37℃ with shaking.
Kanamycin should be omitted when using the cis-repressed pQE-80L series of vectors.
2. Inoculate 50 ml of prewarmed media (with antibiotics) with 2.5 ml of the overnight cultures and grow at 37℃, with vigorous shaking (~300 rpm), until the OD 600 is 0.5–0.7 (approximately 30–60 min).
3. Take a 1-ml sample immediately before induction (noninduced control), pellet cells, and resuspend in 50 ml 1´ SDS -PAGE sample buffer. Freeze the sample at -20℃ until needed for SDS -PAGE.
4. Induce expression by adding IPTG to a final concentration of 1 mM.
5. Grow the cultures for an additional 4–5 hours. Collect a second 1-ml sample (induced control), pellet cells and resuspend in 100 ml 1´SDS-PAGE sample buffer. Freeze until use.
6. Harvest the cells by centrifugation at 4000 ´ g for 20 min.
Protein Expression
1. Resuspend cell pellet in 5 ml of lysis buffer for native purification.
2. Freeze sample in dry ice/ethanol, and thaw in cold water.
Alternatively, add lysozyme to 1mg/ml and incubate on ice for 30 min.
3. Sonicate 6 ´ 10 sec with 10 sec pauses at 200–300 W. Keep lysate on ice at all times.
Use a sonicator with a microtip probe.
4. Centrifuge lysate at 10 000 ´ g at 4℃ for 20–30 min. Decant the supernatant (crude extract A, soluble protein) and save on ice.
5. Resuspend the pellet in 5 ml lysis buffer. This is a suspension of the insoluble matter (crude extract B, insoluble protein).
SDS-PAGE analysis
1. Add 5 ml of 2´ SDS-PAGE sample buffer to 5 ml of crude extracts A & B.
2. Heat these samples, along with the frozen noninduced and induced cell samples at 95℃ for 5 min.
3. Microcentrifuge at 15 000 ´ g for 1 min.
4. Load 20 ml of the noninduced and induced cell samples, and all of the extract samples on a 12% SDS-PAGE gel. Run the gel according to standard procedures.
Interpretation of results
If rior to staining
· A positive control expressing 6xHis-tagged DHFR from pQE-40, if possible
LB medium
Kanamycin stock solution
Ampicillin stock solution
Lysis buffer for purification under native conditions
1´ PAGE sample buffer
2´ PAGE sample buffer
Culture growth
1. Inoculate 10 ml LB medium containing 100 mg/ml ampicillin and 25 mg/ml kanamycin in a 50-ml flask. Grow the cultures overnight at 37℃ with shaking.
Kanamycin should be omitted when using the cis-repressed pQE-80L series of vectors.
2. Inoculate 50 ml of prewarmed media (with antibiotics) with 2.5 ml of the overnight cultures and grow at 37℃, with vigorous shaking (~300 rpm), until the OD 600 is 0.5–0.7 (approximately 30–60 min).
3. Take a 1-ml sample immediately before induction (noninduced control), pellet cells, and resuspend in 50 ml 1´ SDS -PAGE sample buffer. Freeze the sample at -20℃ until needed for SDS -PAGE.
4. Induce expression by adding IPTG to a final concentration of 1 mM.
5. Grow the cultures for an additional 4–5 hours. Collect a second 1-ml sample (induced control), pellet cells and resuspend in 100 ml 1´SDS-PAGE sample buffer. Freeze until use.
6. Harvest the cells by centrifugation at 4000 ´ g for 20 min.
Protein Expression
1. Resuspend cell pellet in 5 ml of lysis buffer for native purification.
2. Freeze sample in dry ice/ethanol, and thaw in cold water.
Alternatively, add lysozyme to 1mg/ml and incubate on ice for 30 min.
3. Sonicate 6 ´ 10 sec with 10 sec pauses at 200–300 W. Keep lysate on ice at all times.
Use a sonicator with a microtip probe.
4. Centrifuge lysate at 10 000 ´ g at 4℃ for 20–30 min. Decant the supernatant (crude extract A, soluble protein) and save on ice.
5. Resuspend the pellet in 5 ml lysis buffer. This is a suspension of the insoluble matter (crude extract B, insoluble protein).
SDS-PAGE analysis
1. Add 5 ml of 2´ SDS-PAGE sample buffer to 5 ml of crude extracts A & B.
2. Heat these samples, along with the frozen noninduced and induced cell samples at 95℃ for 5 min.
3. Microcentrifuge at 15 000 ´ g for 1 min.
4. Load 20 ml of the noninduced and induced cell samples, and all of the extract samples on a 12% SDS-PAGE gel. Run the gel according to standard procedures.
Interpretation of results
If rior to staining
· A positive control expressing 6xHis-tagged DHFR from pQE-40, if possible
