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.
3. Confirm your calculations with the teaching faculty, then prepare your lysis buffer solution.
4. 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.
5. Resuspend each pellet completely in the lysis buffer then transfer the cell suspension to a 2 mL eppendorf tube.
6. Add lysozyme (stock concentration of 50 mg/mL) to each cell suspension such that the final concentration is 300 μg/mL.
7. Incubate in the 4 °C cooler for 1 hr on the nutator.

Part 2: Prepare Ni-NTA affinity column

Keep all buffers on ice when not in use. All spins should be performed at 1000 rcf (3300 rpm) for 1 minute.

1. The following buffers are aliquoted and located at the front laboratory bench:
   • Ni-NTA His-bind resin
   • 1X Ni-NTA Bind Buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 10 mM imidazole)
   • 1X Ni-NTA Wash Buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 20 mM imidazole)
   • 1X Ni-NTA Elute Buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 250 mM imidazole)
   • Note: Two special waste streams should be created for this affinity purification procedure, (1) nickel waste for the 50% slurry, and (2) imidazole waste for the Bind, Wash, and Elute Buffers.
2. Gently mix the Ni-NTA His-bind resin to fully resuspend it, then distribute 400 μL of the resin to each of two 2 mL centrifuge tubes.
   • Label one tube as wild type and the other as mutant.
3. Add 1.6 mL (2 x 800 μL) of 1X Ni-NTA Bind Buffer to the Ni-NTA His-bind resin.
   • Resuspend the resin by pippeting the solution up and down several times (10-15), then centrifuge (see conditions above).
4. Carefully remove the supernatant and discard it in the appropriate waste stream.
5. Equilibrate column with PBS for 30 min at 4 C???

Part 3: Purify FKBP12 protein

1. Retrieve your lysed cell pellets from the 4 °C cooler.
2. 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).
3. 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!
4. Incubate for 30 min in the 4 °C cooler.
5. 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.
6. Check that the supernatent in your sample is clear with little to no 'cloudiness'.
7. Load the supernatent onto the column your prepared in Part 2.
8. Obtain an aliquot of PBS buffer containing 10 mM imidazole from the front laboratory bench.
9. Wash the column by adding 1.5 mL of the PBS buffer containing 10 mM imidazole to the column and invert, then centrifuge at 3.3 rpm for 1 min.
   • Transfer the flow-through to a fresh, well-labeled 2 mL eppendorf tube.
10. Repeat Step #9 a total of three times.
11. Elute your FKBP12 protein by adding 500 μL of PBS buffer containing 250 mM imidazole to the column and invert, then centrifuge at 3.3 rpm for 1 min.
    • Transfer the purified protein sample (the flow-through) to a fresh, well-labeled 1.5 mL eppendorf tube.
12. Give your purified protein solution, the flow-through samples from Step #9, and the column to the teaching faculty for storage until the next laboratory session.

Reagents

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