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Team:Cambridge/Protocols/Protein Purification
From 2011.igem.org
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| - | =Protein Purification= | + | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}} |
| + | __NOTOC__ | ||
| + | |||
| + | ==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 using gravity or centrifugation as described in | ||
| + | Step 2, and gently aspirate the supernatant. | ||
| + | 5. For purification under Denaturing Conditions, add 6 ml of Denaturing | ||
| + | Binding Buffer. | ||
| + | 6. Resuspend the resin by alternately inverting and gently tapping the | ||
| + | column. | ||
| + | 7. Allow the resin to settle using gravity or centrifugation as described in | ||
| + | Step 2, and gently aspirate the supernatant. Repeat Steps 5 through 7. | ||
| + | |||
| + | |||
| + | ====Purification procedure==== | ||
| + | |||
| + | ===Safety=== | ||
| + | All safety measures relating to guanidine hydrochloride in solution from the [https://2011.igem.org/Team:Cambridge/Protocols/Buffers 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. | ||
| + | |||
| + | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | ||
Revision as of 21:56, 20 August 2011
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 using gravity or centrifugation as described in
Step 2, and gently aspirate the supernatant. 5. For purification under Denaturing Conditions, add 6 ml of Denaturing Binding Buffer. 6. Resuspend the resin by alternately inverting and gently tapping the column. 7. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant. Repeat Steps 5 through 7.
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.
