【共享】一个protocol网页
丁香园论坛
680
刚刚看到的，内容不是太多。
http://structure.biochem.queensu.ca/protocols/
感觉有些东西还是不错的，摘录出来：
Purification of 6xHis-Tagged Proteins
Where (Sample) is noted, take an aliquot of the material and boil in SDS-PAGE sample buffer for running on a gel.
1)Inoculate single colony of BL21(DE3) carrying desired plasmid into 20 ml TB + ampicillin (100 g/ml) and culture overnight at 37C
2)Dilute sufficient amount of overnight culture into 1 L TB + ampicillin to give OD600 = 0.2 and culture at 37C for 1 hour (OD600 approx. 0.5-1.0)
3)Add IPTG to 0.1-0.4 mM and culture 4-6 hours at 37C
4)Pellet cells at 6,000 rpm for 5 mins in JA-10 or JA-14 and store at -20C
5)Resuspend cells in 50 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) to which you have added 0.1 % (v/v) Triton X-100, 10 g/ml RNase A (10 mg/ml stock), 5 g/ml DNase I (5 mg/ml stock), 1 mM MgCl2 (1 M stock), 1 mM MnCl2 (1 M stock), and 1 mg/ml lysozyme (sample)
6)Incubate on ice 30 mins; sonicate (10 sec pulses with 30 sec intervals on ice) until becomes partially clarified
7)Centrifuge at 15,000 rpm in JA-20 for 20 mins (sample)
8)Add 5 ml Ni-NTA Agarose (pre-washed with lysis buffer) to supernatant and incubate at 4C with mixing for 30 mins
9)Pour mixture into column and let flow-through drain out (sample)
10) Wash column with 2x50 ml of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0)
11) Elute protein in five 5 ml fractions with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) Do this step by adding 5 ml elution buffer to column, mix thoroughly, and let 5 ml drain from column. Repeat 4 more times.
12)Analyse 3 samples and 5 fractions by SDS-PAGE
13) Pool desired fractions and dialyse against buffer suitable for anion exchange chromatography on FPLC
GST-Fusion Purification and Cleavage
PBS (1 L)
8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4
- dissolve the above in 800 ml ddH2O, adjust pH to 7.3 with HCl, then bring to 1L
1.Resuspend cells in 50 ml GST lysis buffer (PBS + 0.1% Triton X-100, 1 mM DTT, 5ug/ml DNAse I, 10 ug/ml RNAse A, 1 mg/ml lysozyme, and 1 mM PMSF)
2.Incubate resuspended cells on ice for 30 minutes.
3.Meanwhile, begin equilibrating a 5mL GST-Trap column (Amersham-Pharmacia) by pumping GST through it with a peristaltic pump. Pump at least 3 column volumes (15 mL) through the column.
4.Sonicate cells for one 20-second cycle. (This should be enough sonication since we used lysozyme above. Over-sonicating disrupts the GST tag and makes it so that the protein will not bind the column!
5.Centrifuge at 15 000 rpm (JA-20 rotor) for 20 minutes.
6.Collect supernantant, and load it very slowly on to the equilibrated GST-Trap column. Pump at 0.5 mL/min or less.
7.Wash column extensively with 150 mL PBS. The pump can be run at more than 1 mL/min at this stage.
8.Mix 40 units of thrombin in 5 mL PBS. Pump this 5 mL thrombin solution in to the column, then shut down the pump. Allow thrombin to cleave the fusion protein overnight at room temperature. (If you have evidence that your protein degrades at room temperature, perform this step in the cold room).
9.Elute you protein of interest with 4 x 5 mL fractions of PBS. These fractions will contain your protein without GST attached.
10.Elute GST from the column in a 15 mL fraction of GST elution buffer (50mM Tris-HCl, pH8.0 with 10 mM reduced glutathione). This fraction will contain GST alone plus any GST-fusion that was not cleaved by thrombin above.
11.Run gels of all fractions to determine purity, etc. Pool and concentrated as desired.
另外一个PCR引物设计教程，认为写得很清楚：
http://structure.biochem.queensu.ca/protocols/cloning.pdf
http://structure.biochem.queensu.ca/protocols/
感觉有些东西还是不错的，摘录出来：
Purification of 6xHis-Tagged Proteins
Where (Sample) is noted, take an aliquot of the material and boil in SDS-PAGE sample buffer for running on a gel.
1)Inoculate single colony of BL21(DE3) carrying desired plasmid into 20 ml TB + ampicillin (100 g/ml) and culture overnight at 37C
2)Dilute sufficient amount of overnight culture into 1 L TB + ampicillin to give OD600 = 0.2 and culture at 37C for 1 hour (OD600 approx. 0.5-1.0)
3)Add IPTG to 0.1-0.4 mM and culture 4-6 hours at 37C
4)Pellet cells at 6,000 rpm for 5 mins in JA-10 or JA-14 and store at -20C
5)Resuspend cells in 50 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) to which you have added 0.1 % (v/v) Triton X-100, 10 g/ml RNase A (10 mg/ml stock), 5 g/ml DNase I (5 mg/ml stock), 1 mM MgCl2 (1 M stock), 1 mM MnCl2 (1 M stock), and 1 mg/ml lysozyme (sample)
6)Incubate on ice 30 mins; sonicate (10 sec pulses with 30 sec intervals on ice) until becomes partially clarified
7)Centrifuge at 15,000 rpm in JA-20 for 20 mins (sample)
8)Add 5 ml Ni-NTA Agarose (pre-washed with lysis buffer) to supernatant and incubate at 4C with mixing for 30 mins
9)Pour mixture into column and let flow-through drain out (sample)
10) Wash column with 2x50 ml of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0)
11) Elute protein in five 5 ml fractions with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) Do this step by adding 5 ml elution buffer to column, mix thoroughly, and let 5 ml drain from column. Repeat 4 more times.
12)Analyse 3 samples and 5 fractions by SDS-PAGE
13) Pool desired fractions and dialyse against buffer suitable for anion exchange chromatography on FPLC
GST-Fusion Purification and Cleavage
PBS (1 L)
8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4
- dissolve the above in 800 ml ddH2O, adjust pH to 7.3 with HCl, then bring to 1L
1.Resuspend cells in 50 ml GST lysis buffer (PBS + 0.1% Triton X-100, 1 mM DTT, 5ug/ml DNAse I, 10 ug/ml RNAse A, 1 mg/ml lysozyme, and 1 mM PMSF)
2.Incubate resuspended cells on ice for 30 minutes.
3.Meanwhile, begin equilibrating a 5mL GST-Trap column (Amersham-Pharmacia) by pumping GST through it with a peristaltic pump. Pump at least 3 column volumes (15 mL) through the column.
4.Sonicate cells for one 20-second cycle. (This should be enough sonication since we used lysozyme above. Over-sonicating disrupts the GST tag and makes it so that the protein will not bind the column!
5.Centrifuge at 15 000 rpm (JA-20 rotor) for 20 minutes.
6.Collect supernantant, and load it very slowly on to the equilibrated GST-Trap column. Pump at 0.5 mL/min or less.
7.Wash column extensively with 150 mL PBS. The pump can be run at more than 1 mL/min at this stage.
8.Mix 40 units of thrombin in 5 mL PBS. Pump this 5 mL thrombin solution in to the column, then shut down the pump. Allow thrombin to cleave the fusion protein overnight at room temperature. (If you have evidence that your protein degrades at room temperature, perform this step in the cold room).
9.Elute you protein of interest with 4 x 5 mL fractions of PBS. These fractions will contain your protein without GST attached.
10.Elute GST from the column in a 15 mL fraction of GST elution buffer (50mM Tris-HCl, pH8.0 with 10 mM reduced glutathione). This fraction will contain GST alone plus any GST-fusion that was not cleaved by thrombin above.
11.Run gels of all fractions to determine purity, etc. Pool and concentrated as desired.
另外一个PCR引物设计教程，认为写得很清楚：
http://structure.biochem.queensu.ca/protocols/cloning.pdf
