20.109(S17):Purification of induced protein (Day2)
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Contents
Introduction
Protocols
Part 1: Lyse BL21(DE3)pLysS pRSETb_FKBP12 cells
- 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.
- 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.
- Confirm your calculations with the teaching faculty, then prepare your lysis buffer solution.
- Weigh each cell pellet using the balance and add 1 mL of lysis buffer per g of cell pellet.
- Note: the weight of the 50 mL conical tube is XX g.
- Resuspend each pellet completely in the lysis buffer then transfer the cell suspension to a 2 mL eppendorf tube.
- Add lysozyme (stock concentration of 50 mg/mL) to each cell suspension such that the final concentration is 300 μg/mL.
- Incubate in the 4 °C cooler for 1 hr on the nutator.
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 |
Part 2: Purify FKBP12 protein
- Retrieve your lysed cell pellets from the 4 °C cooler.
- Transfer XX from each tube into labeled 1.5 mL eppendorf tubes and give the aliquots to the teaching faculty.
- You will use these aliquots during the next laboratory session to examine protein yield using polyacrylamide gel electrophoresis (PAGE).
- Add XX μL MgCl2 and 10 μL of DNase to the tube that contains the IPTG-induced sample.
- You will only complete the purification protocol for the IPTG-induced sample!
- Incubate for 30 min in the 4 °C cooler.
- Centrifuge your sample for 30 min at 16,000 rcf in the 4 °C cold room.
- Alert the teaching faculty when you are ready to centrifuge your sample and you will be escorted to the cold room.
- Check that the supernatent in your sample is clear with little to no 'cloudiness'.
- Load the supernatent
Reagents
Next day: Evaluation of purified protein
Previous day: In silico cloning and induction of protein expression