Mini-Expression of 6-His Fusion Proteins
Mini-Expression of 6-His Fusion Proteins
1. Grow ON of single colony in 5 ml of LB-AMP at 37 °C.
2. In morning, dilute 1/50 into 5 ml fresh LB-AMP and grow at 37 °C.
3. Grow to OD 600 of 0.5 to 0.7- Do not overgrow.
4. When it reaches the correct OD, save 1 ml of the culture on ice (=uninduced
sample).
5. To the rest of the culture, add IPTG to 0.5 mM final to induce expression.
6. Grow for an additional 2 to 4 hrs at 37 °C.
7. Read OD 600 of the induced cultures.
8. Save 1 ml of the induced cultures (=induced sample).
9. Spin down 1 ml of both the uninduced and induced samples at full speed for 1
min in the microfuge.
10. Resuspend in sample buffer using the ratio of 50 μl of SB per 0.5 OD units. This
way all samples will have about the same amount of protein.
11. Run out on SDS-PAGE running about 5-8 μl/lane and look for bands in the
induced culture relative to the uninduced culture.
To test solubility of fusion proteins, follow procedure exactly as above through step 9
then continue below.
12. Resuspend cells in 100 μl of lysis buffer ((50 mM Pi, pH 8.0; 300 mM NaCl; 0.1 %
Tween 20; 10 mM Imidazole + 1 mM PMSF + 0.5 mg/ml lysozyme)).
13. Freeze in liquid nitrogen and thaw on ice.
14. Sonicate 10 sec each with a small tip sonicator.
15. Spin 10’ in 4 °C microfuge.
16. Take sup to a fresh tube and resuspend pellet in 100 μl of lysis buffer (pellet may
need to be sonicated gently to get it to go into solution).
17. Take 7.5 μl of sample and 7.5 μl of SB, boil and run out 10 μl on SDS-PAGE.
Trouble shooting and variables to test: to increase solubility:
There are several options to try to increase the solubility or expression of the fusion
proteins.
1. Try a bacterial expression strain with tighter stringency such as BL21(DE3)pLysS.
2. Lower the temperature of induction to 25 °C rather than 37 °C.
3. Use lower amounts of IPTG for induction (0.01 mM to 0.1 mM).
4. Induce at a higher OD (about 1)’
5. Add higher salt (0.5 M NaCl) to lysis buffer.
6. Add 2 M urea to the lysis buffer.
7. Don’t grow ON before induction- innoculate colony directly into media in the
morning.
1. Grow ON of single colony in 5 ml of LB-AMP at 37 °C.
2. In morning, dilute 1/50 into 5 ml fresh LB-AMP and grow at 37 °C.
3. Grow to OD 600 of 0.5 to 0.7- Do not overgrow.
4. When it reaches the correct OD, save 1 ml of the culture on ice (=uninduced
sample).
5. To the rest of the culture, add IPTG to 0.5 mM final to induce expression.
6. Grow for an additional 2 to 4 hrs at 37 °C.
7. Read OD 600 of the induced cultures.
8. Save 1 ml of the induced cultures (=induced sample).
9. Spin down 1 ml of both the uninduced and induced samples at full speed for 1
min in the microfuge.
10. Resuspend in sample buffer using the ratio of 50 μl of SB per 0.5 OD units. This
way all samples will have about the same amount of protein.
11. Run out on SDS-PAGE running about 5-8 μl/lane and look for bands in the
induced culture relative to the uninduced culture.
To test solubility of fusion proteins, follow procedure exactly as above through step 9
then continue below.
12. Resuspend cells in 100 μl of lysis buffer ((50 mM Pi, pH 8.0; 300 mM NaCl; 0.1 %
Tween 20; 10 mM Imidazole + 1 mM PMSF + 0.5 mg/ml lysozyme)).
13. Freeze in liquid nitrogen and thaw on ice.
14. Sonicate 10 sec each with a small tip sonicator.
15. Spin 10’ in 4 °C microfuge.
16. Take sup to a fresh tube and resuspend pellet in 100 μl of lysis buffer (pellet may
need to be sonicated gently to get it to go into solution).
17. Take 7.5 μl of sample and 7.5 μl of SB, boil and run out 10 μl on SDS-PAGE.
Trouble shooting and variables to test: to increase solubility:
There are several options to try to increase the solubility or expression of the fusion
proteins.
1. Try a bacterial expression strain with tighter stringency such as BL21(DE3)pLysS.
2. Lower the temperature of induction to 25 °C rather than 37 °C.
3. Use lower amounts of IPTG for induction (0.01 mM to 0.1 mM).
4. Induce at a higher OD (about 1)’
5. Add higher salt (0.5 M NaCl) to lysis buffer.
6. Add 2 M urea to the lysis buffer.
7. Don’t grow ON before induction- innoculate colony directly into media in the
morning.
You can find IPTG Buffer at Alkali Scientific. They also have other reagents and lab chemicals.
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