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Team:Cambridge/Protocols/Protein Purification
From 2011.igem.org
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:# Pipette 2 ml of the resin into a 10-ml Purification Column. Allow the resin to settle completely by gravity (5-10 minutes) before carefully aspirating the supernatant. | :# Pipette 2 ml of the resin into a 10-ml Purification Column. Allow the resin to settle completely by gravity (5-10 minutes) before carefully aspirating the supernatant. | ||
:# Add 6 ml of sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column. | :# Add 6 ml of sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column. | ||
| - | : Allow the resin to settle using gravity | + | :# Allow the resin to settle again using gravity, and gently aspirate the supernatant. |
| - | + | :# Add 6 ml of Denaturing Binding Buffer and resuspend the resin by alternately inverting and gently tapping the column. | |
| - | + | :# Allow the resin to settle using gravity, and gently aspirate the supernatant. Repeat Steps 5 and 6. | |
| - | Binding Buffer | + | |
| - | + | ||
| - | column. | + | |
| - | + | ||
| - | + | ||
| - | + | ||
====Purification procedure==== | ====Purification procedure==== | ||
| + | :# | ||
===Safety=== | ===Safety=== | ||
Revision as of 22:36, 20 August 2011
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Protein Purification
A his-trap mechanism was used to isolate reflectin from the lysate produced after performing an [inclusion body prep]. The protocol below was adapted from [http://tools.invitrogen.com/content/sfs/manuals/xprpur_man.pdf here].
Theory
Practice
Preparing the column
- When taking his-trap resin out of storage, the resin is likely to have precipitated from its storage buffer. Resuspend the resin in its bottle by inverting and gently
tapping the bottle repeatedly.
- Pipette 2 ml of the resin into a 10-ml Purification Column. Allow the resin to settle completely by gravity (5-10 minutes) before carefully aspirating the supernatant.
- Add 6 ml of sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column.
- Allow the resin to settle again using gravity, and gently aspirate the supernatant.
- Add 6 ml of Denaturing Binding Buffer and resuspend the resin by alternately inverting and gently tapping the column.
- Allow the resin to settle using gravity, and gently aspirate the supernatant. Repeat Steps 5 and 6.
Purification procedure
Safety
All safety measures relating to guanidine hydrochloride in solution from the buffer protocol apply here when dealing with GLB. In addition, all consumables and liquid waste that may have come into contact with the bacteria must be autoclaved before disposal.
