Protocol P3
Jun 24, 2009
PROTOCOL FOR EXTRACTION AND PURIFICATION OF SOLUBLE PROTEINS
1. Overnight culture - Inoculate 5ml of LB medium with cells containing your gene of interest.
i)
Incubate the culture at 370C for overnight.
2. Scaling up the culture - Next day, transfer 0.5 -1ml from overnight culture to a larger volume
(250-300ml) culture and grow at 370C for C-41 or BL-21 cells, or 300C for Arctic Express cells.
3. Induction - When the O.D. reaches a value of 0.6 - 0.8 induce the cells with 0.4mM IPTG for
Arctic Express and C-41 cells, and grow the culture at lower temperature optimized for
expression of your protein of interest (130C in this case).
4. Harvesting the cells - Harvest the cells after 16 - 18 hrs of induction.
For harvesting,
i)
Use centrifuge tubes with a capacity of 250 - 300ml of volume.
ii)
Weigh the centrifuge tubes before putting cell culture in them.
iii)
Centrifuge for 10 minutes at 6 degrees at 10,000g.
iv)
Discard the supernatant.
v)
Weigh the centrifuge tubes with cell pellet in them.
vi)
Determine the weight of the cell pellet.
Meanwhile, equilibration of the column – You may start equilibrating the column by applying
10 - 15 volumes of 1X binding buffer.
5. Lysis of the cells - Add 10ml of extraction buffer (1X binding buffer/lysis buffer) for each gram of
cell-pellet’s fresh-weight.
For making extraction buffer,
i)
Use the lysis buffer (1X Binding Buffer – check the recipe at the end of this
document).
ii)
Add following reagents to the 1X Binding Buffer for facilitating lysis of the cells a) β-mercapto ethanol (2-MCE)
- 20mM
b) Phenyl methyl sulfonyl fluoride (PMSF)
- 0.1mg/ml
c) Triton-X
- 0.1% [v/v]
d) EDTA
- 1mM
iii)
Re-suspend the pellet in the extraction buffer.
6. Lysis by lysozyme - Add lysozyme to the suspended pellet, in order to lyse the bacterial cell wall.
i)
Concentration of lysozyme – 5KU per gram of cell pellet.
ii)
Incubate for 20’ at 40C.
7. Lysis by sonication - Sonicate the samples six - seven times, with an exposure to ultrasonic
waves for about 10 seconds followed by cooling of the sample for about a minute.
Shivani Garg
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Protocol P3
Jun 24, 2009
i)
Withdraw a 200μl sample (whole cell extract sample) for analysis by SDS PAGE.
8. Separation of soluble fraction from insoluble one - Centrifuge the samples for 30’ at 60C at
15,000g.
i)
Collect the supernatant.
ii)
Withdraw a 200μl sample (soluble fraction sample) for analysis by SDS PAGE.
9. Sterilization of the soluble fraction - Sterilize the soluble fraction by filter sterilization and store
in a falcon tube.
10. Application of protein to the column - Apply the soluble fraction to the column.
i)
Collect the flow-through in a new falcon tube.
ii)
Bradford test - Set up a Bradford test in a 96 well micro-titer plate.
a) Add 200μl of diluted Bradford dye to each of the wells.
b) As the sample is flowing out of the column, monitor the decrease in
protein content by collecting 1-2 μl from the tip of the column and
putting it into the well containing Bradford dye.
c) If the micro-titer plate wells show intense blue color, re-apply the
flow through to the column to increase the protein binding (Step
11).
iii)
Withdraw a 200ul sample (first flow-through sample) for analysis by SDS PAGE.
11. Re-application of the protein sample - Depending on your Bradford test results, re-apply the
flow-through to the column.
i)
You may re-apply the sample for the third time if necessary.
12. Washing the column with binding buffer - Wash the column with 5-10 volumes of 1X binding
buffer.
i)
Withdraw 1ml sample for SDS PAGE.
ii)
Monitor the color change of the wells using the Bradford test. If the wells
progressively start to show color change from blue to red, it means no more
protein is being washed out. This serves as an indication to move to washing
buffer with higher concentration of imidazole.
13. First wash of the column with wash buffer - Add wash buffer 1.
i)
Use falcon tubes for collecting the flow-through for each of the wash buffer that
you apply.
ii)
Also monitor the color change in micro-titer plate wells. If it changes from blue
to red, move to next wash buffer with higher imidazole concentration.
Shivani Garg
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Protocol P3
Jun 24, 2009
14. Subsequent washing with buffers having higher imidazole concentration –
If this is your first protein run through the column, it is advisable to try a series of wash buffers
to determine where your protein of interest elutes out.
i)
Try different wash buffers with increasing imidazole concentrations, for example
from 20mM, 40mM, 60mM, 80mM, 100mM, 120mM, 200mM to 250mM.
ii)
Collect the flow-through from each of the wash buffers in falcon tubes.
iii)
Keep on monitoring if the protein is being eluted out of the column using
Bradford test in micro-titer plate. As soon as you see no color change in the
Bradford dye, switch to a buffer with higher imidazole concentration.
iv)
Withdraw 1ml sample from each of the flow-through fractions for SDS PAGE
analysis.
If you already know the concentrations of imidazole at which contaminants (let’s call it wash
buffer C) and your protein (let’s call it wash buffer P) elute out respectively, then
i)
After washing with 1X binding buffer, use the wash buffer C to wash out all
contaminants.
The volume of the wash buffer C would be dictated by the Bradford test results
that you get while monitoring the eluent from the sample. If you no longer see
much color change from red to blue, it indicates that most of your
contaminating proteins have been washed away.
You can now move to wash buffer P.
ii)
Use wash buffer P for washing your protein out of the column.
Again the volume for wash buffer P would be dictated by monitoring color
change of Bradford dye in the micro-titer plate.
15. Analysis by SDS PAGE - Analyze your protein elution fractions by SDS PAGE.
16. His-tag removal - Carry out His-tag cleavage.
17. Analysis by SDS PAGE - Analyze the cleavage by SDS-PAGE.
Recipe for 8X Binding Buffer
40 mM imidazole
4 M NaCl
160 mM Tris-HCl, pH8.0
Shivani Garg
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