Protocol
A) Induction of Bacterial culture.
1) Take 50ml of LB broth and add the required antibiotic in the suitable concentration and 2ml of the culture.
2) Incubate at 370C at 220 rmp, till the OD at 600nm reaches 0.8-1.
3) Once the OD is reached add 0.2mM IPTG to the above culture.
5) Incubate at 370C at 220 rmp for 1-1:30 hrs.
4) Store at 40C till the extraction process.
B) Extraction of Protein from the Induced Culture.
1) Transfer 50ml of induced and uninduced (as negative control) in 50ml tube.
2) Centrifuge at 14,000rpm for 1min or 5,000rpm for 5mins at 40C.
3) Completely decant the supernatant and dislodged the pellet to make a single cell suspension.
4) Take 10ml of Binding Buffer and to that add 10ul of Triton-X100 + 10ul of cocktail of Protease Inhibitor.
5) Add 2ul of the above mix to the single cell suspension.
6) Vortex properly for about 10-20secs.
7) Transfer the above solution to a eppendrof.
8) Dis rupture the cells using sonicator at 35 amplitude for 1min with pulse on for 1sec and pulse off for 1sec.
9) After sonication, incubate the on ice for 15mins.
10) Centrifuge for 30mins at 12,000rpm, 40C.
11) Transfer the supernatant on another eppendroff, and store the pellet at -800.
12) Load the supernatant on the column bases on the property of the protein need to be isolated and collect the flow through.
13) Add 10 volumes of Binding buffer based on the column height.
14) Wash with wash buffer and collect the wash.
15) Pass different concentrations of Imidazole Elution Buffer and collect elution.
16) Run all the flow through and elution on SDS-PAGE.
C) Preparation of the Column
1) Take a 5ml syringe and seal one end of the using glass wool.
2) Add some water to the column and over that slowly from the sides add HisPur Cobalt Resin.
3) Fill the column with resin up to 1ml marks in the syringe.
4) Wash the column with 3 ml of water.
5) Equilibrate the column with 3 volumes of Binding buffer based on the amount of resin added.
6) The column is ready to be used.
7) Once all the samples are run, we should pass 100mM EDTA through the column. This process is called as stripping of the column and the column is ready for the next use and after a repeated use for a long time. we can again charge the column using cobalt.
8) Seal both the ends of the column using parafilm. and store at 40C till further used.
NOTE: At any point during the experiment the column should not be dry.
Solutions
1) Binding Buffer (pH 7.8)
20mM Sodium Phosphate
50mM NaCl
2) Wash Buffer (pH 6)
20mM Sodium Phosphate
50mM NaCl
3) Imidazole Elution Buffer (pH 6)
20mM Sodium Phosphate
50mM NaCl
100mM, 200mM, 300mM, 400mM and 500mM Imidazole
4) 20mM Sodium Phosphate
pH Volume of Na2HPO4(ml) Volume of NaH2PO4(ml)
7.8 89.6 10.4
6.0 12.0 88.0
Dilute the 1M stock solution to 1liters with distil water.
A) Induction of Bacterial culture.
1) Take 50ml of LB broth and add the required antibiotic in the suitable concentration and 2ml of the culture.
2) Incubate at 370C at 220 rmp, till the OD at 600nm reaches 0.8-1.
3) Once the OD is reached add 0.2mM IPTG to the above culture.
5) Incubate at 370C at 220 rmp for 1-1:30 hrs.
4) Store at 40C till the extraction process.
B) Extraction of Protein from the Induced Culture.
1) Transfer 50ml of induced and uninduced (as negative control) in 50ml tube.
2) Centrifuge at 14,000rpm for 1min or 5,000rpm for 5mins at 40C.
3) Completely decant the supernatant and dislodged the pellet to make a single cell suspension.
4) Take 10ml of Binding Buffer and to that add 10ul of Triton-X100 + 10ul of cocktail of Protease Inhibitor.
5) Add 2ul of the above mix to the single cell suspension.
6) Vortex properly for about 10-20secs.
7) Transfer the above solution to a eppendrof.
8) Dis rupture the cells using sonicator at 35 amplitude for 1min with pulse on for 1sec and pulse off for 1sec.
9) After sonication, incubate the on ice for 15mins.
10) Centrifuge for 30mins at 12,000rpm, 40C.
11) Transfer the supernatant on another eppendroff, and store the pellet at -800.
12) Load the supernatant on the column bases on the property of the protein need to be isolated and collect the flow through.
13) Add 10 volumes of Binding buffer based on the column height.
14) Wash with wash buffer and collect the wash.
15) Pass different concentrations of Imidazole Elution Buffer and collect elution.
16) Run all the flow through and elution on SDS-PAGE.
C) Preparation of the Column
1) Take a 5ml syringe and seal one end of the using glass wool.
2) Add some water to the column and over that slowly from the sides add HisPur Cobalt Resin.
3) Fill the column with resin up to 1ml marks in the syringe.
4) Wash the column with 3 ml of water.
5) Equilibrate the column with 3 volumes of Binding buffer based on the amount of resin added.
6) The column is ready to be used.
7) Once all the samples are run, we should pass 100mM EDTA through the column. This process is called as stripping of the column and the column is ready for the next use and after a repeated use for a long time. we can again charge the column using cobalt.
8) Seal both the ends of the column using parafilm. and store at 40C till further used.
NOTE: At any point during the experiment the column should not be dry.
Solutions
1) Binding Buffer (pH 7.8)
20mM Sodium Phosphate
50mM NaCl
2) Wash Buffer (pH 6)
20mM Sodium Phosphate
50mM NaCl
3) Imidazole Elution Buffer (pH 6)
20mM Sodium Phosphate
50mM NaCl
100mM, 200mM, 300mM, 400mM and 500mM Imidazole
4) 20mM Sodium Phosphate
pH Volume of Na2HPO4(ml) Volume of NaH2PO4(ml)
7.8 89.6 10.4
6.0 12.0 88.0
Dilute the 1M stock solution to 1liters with distil water.
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