Difference between revisions of "20.109(S17):Purification of induced protein (Day2)"
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Noreen Lyell (Talk | contribs) (→Part 2: Prepare Ni-NTA affinity column) |
Noreen Lyell (Talk | contribs) (→Part 3: Purify FKBP12 protein) |
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#Transfer XX from each tube into labeled 1.5 mL eppendorf tubes and give the aliquots to the teaching faculty. | #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). | #*You will use these aliquots during the next laboratory session to examine protein yield using polyacrylamide gel electrophoresis (PAGE). | ||
− | #Add XX μL | + | #Add XX μL MgCl<sub>2</sub> 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! | #*You will only complete the purification protocol for the IPTG-induced sample! | ||
#Incubate for 30 min in the 4 °C cooler. | #Incubate for 30 min in the 4 °C cooler. | ||
Line 76: | Line 76: | ||
#*Alert the teaching faculty when you are ready to centrifuge your sample and you will be escorted to the 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'. | #Check that the supernatent in your sample is clear with little to no 'cloudiness'. | ||
− | #Load the supernatent | + | #Load the supernatent onto the column your prepared in Part 2. |
+ | #Obtain an aliquot of PBS buffer containing 10 mM imidazole from the front laboratory bench. | ||
+ | #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. | ||
+ | #Repeat Step #9 a total of three times. | ||
+ | #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. | ||
+ | #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== | ==Reagents== |
Revision as of 00:50, 4 January 2017
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: 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.
- 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.
- 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.
- 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).
- Carefully remove the supernatant and discard it in the appropriate waste stream.
- Equilibrate column with PBS for 30 min at 4 C???
Part 3: 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 onto the column your prepared in Part 2.
- Obtain an aliquot of PBS buffer containing 10 mM imidazole from the front laboratory bench.
- 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.
- Repeat Step #9 a total of three times.
- 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.
- 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
Next day: Evaluation of purified protein
Previous day: In silico cloning and induction of protein expression