Difference between revisions of "20.109(S17):Purification of induced protein (Day2)"

Introduction

Protocols

Part 1: Lyse BL21(DE3)pLysS pRSETb_FKBP12 cells

1. Retrieve your BL21(DE3)pLysS pRSETb_FKBP12 cell pellet from the front laboratory bench and leave them on your bench to thaw.
   • You will also obtain a cell pellet of uninduced cells that was prepared by the teaching faculty as a control to examine the IPTG induction step.
2. Prepare 3 mL of lysis buffer.
   • Use the information in the table below to calculate the volume of each stock reagent that is needed to prepare the lysis buffer with the specified final concentrations:
   • {| border="1"

! Stock reagent ! Final concentration of stock reagent in lysis buffer ! Volume of stock reagant |- | 1 M Tris (pH = 7) | 50 mM | |- | 1 M NaCl | 150 mM | |- | 40% glycerol | 10% | |- | 1 M DTT | 1 mM | |- | 1 M AEBSF | 1 mM | |- | H₂O | add for a total of 3 mL of lysis buffer | |}

1. Confirm your calculations with the teaching faculty, then prepare your lysis buffer solution.
2. Weigh each cell pellet using the balance and add 1 mL of lysis buffer per g of cell pellet.
   • Note: the weight of the 2 mL eppendorf tube is 1.1g.
3. Resuspend each pellet completely in the lysis buffer.
4. Add lysozyme (stock concentration of 50 mg/mL) to each cell resuspension such that the final concentration is 300 μg/mL.
5. Incubate in the 4 °C cooler for 1 hr on the nutator.

Part 2: Purify FKBP12 protein

Reagents

Navigation links

Next day: Evaluation of purified protein