Team:Cambridge/Protocols/Protein Purification

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

1. 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.

1. 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.
2. Add 6 ml of sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column.
3. Allow the resin to settle again using gravity, and gently aspirate the supernatant.
4. Add 6 ml of Denaturing Binding Buffer and resuspend the resin by alternately inverting and gently tapping the column.
5. Allow the resin to settle using gravity, and gently aspirate the supernatant. Repeat Steps 5 and 6.

Purification procedure

1. Clamp a prepared purification column vertically at a convenient height for pipetting into. Add 8 ml lysate from an [inclusion body prep].

2. Bind for 15–30 minutes at room temperatureby inverting up and down to keep the resin suspended in the lysate solution. Settle the resin by gravity and carefully aspirate the supernatant. 3. Wash the column with 4 ml Denaturing Binding Buffer by resuspending the resin and rocking for two minutes. Settle the resin by gravity, and carefully aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE analysis. Repeat this step one more time. 4. Wash the column with 4 ml Denaturing Wash Buffer, pH 6.0 supplied in the kit by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 × g), and carefully aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE analysis. Repeat this step one more time. 5. Wash the column with 4 ml Denaturing Wash Buffer pH 5.3 (see recipe on previous page) by resuspending the resin and rocking for 2 minutes. Settle the resin by gravity or low speed centrifugation (800 × g), and carefully aspirate the supernatant. Save supernatant at 4°C for SDS- PAGE analysis. Repeat this step once more for a total of two washes with Denaturing Wash Buffer pH 5.3. 6. Clamp the column in a vertical position and snap off the cap on the lower end. Elute the protein by adding 5 ml Denaturing Elution Buffer supplied with the kit. Collect 1 ml fractions and monitor the elution by taking OD280 readings of the fractions. Pool the fractions that contain the peak absorbance and dialyze against 10 mM Tris, pH 8.0, 0.1% Triton X- 100 overnight at 4°C to remove the urea. Concentrate the dialyzed material by any standard method (i.e., using 10,000 MW cut-off, low

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.