Continuing to Purify Asc Hb with Ni(protoporphyrin IX)
- A new PD-10 Desalting column was used to continue the purification today:
- A PD-10 Desalting column was prepared with one column volume of dH2O was run over the column, and two column volumes of 25mM Tris, pH 8.3 run over the column.
- 2.5mL of the dialyzed Reconstituted Asc Hb was then run over the column.
- The protein was eluted off with 3.5mL of 25mM Tris, pH 8.3.
- The loading and elution of the protein was repeated many times.
- The protein was then purified over a HiTrap Q FF column on an FPLC:
- Turn on and synchronize pumps, then turn on computer and open software.
- Run PumpWashBasic in dH2O.
- Set the flow rate at 5mL/min, this will be the flow rate for all subsequent steps in this procedure.
- Run 25mL dH2O on Position1ByPass.
- Run 25mL dH2O on Position3 (which is over the column).
- the column is a HiTrap Q FF column by GE Healthcare Life Sciences
- Run 25mL 25mM Tris, 50mM NaCl, pH 8.3 buffer on Position1ByPass.
- Run 25mL 25mM Tris, 50mM NaCl, pH 8.3 buffer over column.
- Inject all of the protein that came off of the PD-10 Desalting column (approx 75mL) onto the column.
- Run 25mL 25mM Tris, 50mM NaCl, pH 8.3 buffer over column to wash the column.
- Run a gradient of NaCl from 50mM to 0.5M over 30 minutes over the column in 25mM Tris, pH 8.3.
- Run 25mL of 25mM Tris, 1M NaCl, pH 8.3 buffer over the column to remove any residual protein.
- Run 25mM Tris, 50mM NaCl, pH 8.3 buffer over the column.
- Run 25mL dH2O over the column and on Position1ByPass to clean the column and tubes.
- Run 25mL 20% ethanol over the column and on Position1ByPass to clean and store the column and tubes.
The graph produced by the FPLC during the purification. It shows that most of the protein came off starting in fraction 11.
Concentrating Asc Hb with Ni(protoporphyrin IX)
A Jumbosep™ Centrifugal Device was used to concentrate the protein:
- Tubes 11-22 (60mL) were poured into the Jumbosep™ Centrifugal Device.
- The device was then spun at 3000g for 30 minutes at 4°C.
- The flowthrough was discarded (looked clear) and there appeared to be about 10mL of concentrated protein in the device.
- Tubes 23-30 (40mL) were poured into the Jumbosep™ Centrifugal Device with the already concentrated protein.
- Again the device was then spun at 3000g for 30 minutes at 4°C.
- The device was then spun at 3000g for 10 minutes at 4°C.
- About 6.5mL of concentrated protein remained, which was saved and stored at 4°C.
Cutting with HindIII for Cloning
This was done with the PCR clones (wt Asc Hb and M8S/M33S/M103S Asc Hb) that were gel-purified on 07/02/12. This will be done in NEBuffer4 instead of 2 because the BamHI has reduced star activity in this buffer, and maybe star activity could be affecting the cuts that lead to poor ligations.
- For the each PCR clone, mix 35μL of DNA with 5μL of 10x NEBuffer4, 5μL of sterile dH2O, and 5μL of HindIII enzyme.
- Incubate for two hours at 37°C.
- Incubate for 20 minutes at 65°C.
- Store at -20°C.
Looking at Sequences
Sequences finally came back and here are the successful colonies (since the minipreps are started from a single colony, I label glycerol stocks and DNA with a colony number):
- M8S/M33L/M103S: colony 3 has the correct sequence
- M8S/M33S/M103S/A71M: colonies 2 and 3 have the correct sequence
- M103S: Colonies 1 and 3 have the correct sequence
Running a DNA Gel
- A 2% agarose gel was made by mixing 0.5g of agarose with 25mL TAE buffer. This mixture was heated in the microwave for 42 seconds and then poured into a gel chamber with a comb inserted.
- When the gel had hardened the comb was removed and 5μL of pre-mixed DNA ladder was loaded into the first lane.
- 25μL of the PCR cloned wildtype Asc Hb 06/29/12 with 5μL of 6x loading dye was loaded into the first wide lane.
- A lane was skipped.
- 5μL of the wild-type HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the next lane.
- 5μL of the M8S/M33S/M103S HindIII cut Asc Hb with 1μL of 6x loading dye was loaded into the next lane.
- 25μL of the PCR cloned M8S/M33S/M103S Asc Hb 06/29/12 with 5μL of 6x loading dye was loaded into the second wide lane.
- The gel was run at 200V until the dye band had moved about 3/4 of the way down the gel.
- The gel was then stained in ethidium bromide for about 30 minutes.
Beginning the The Wizard® SV Gel and PCR Clean-Up System Protocol
The full protocol can be found here. This was done for the two large bands on the gel run today (PCR clones from 06/29/12)
- Two 1.5mL microcentrifuge tubes were weighed and their weights were recorded.
- Tube for wild-type Asc Hb: 1.03g
- Tube for triple mutant Asc Hb: 1.04g
- The large bands on the gel were cut out of the gel with a razor blade (additional safety required with UV light).
- The bands were placed into the microcentrifuge tubes and the tubes were re-weighed.
- Tube for wild-type Asc Hb: 1.21g
- Tube for triple mutant Asc Hb: 1.19g
- The microcentrifuge tubes will be stored at 4°C overnight.
Making Wildtype Asc Hb "Apo"
This procedure was done twice, with two 20mL portions of the wildtype Asc Hb:
- 230μL of 1M HCl was added to 20mL of wildtype Asc Hb in 25mM Tris (pH 8).
- This step hopefully lowered the pH of the protein to 2.3
- In the cold room 20mL of 2-butanone was added to the protein and the protein was shaken vigorously for 30 seconds.
- The solution sat for about five minutes and then a clear seperation was seen (the top half was pink and the bottom half was almost clear). The top half (the heme in 2-butanone) was removed by a Pasteur pipette.
- 20mL more of 2-butanone was added to the protein, shaken vigorously for 30 seconds, allowed to sit for five minutes, and then when separation was seen the top half was removed by a Pasteur pipette.
- The previous step was repeated one more time.
- The protein layer was then transferred to dialysis tubing.
- The dialysis tubing was placed in 2L of 10mM NaHCO3.
- Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
- The protein was dialyzed overnight (with stirring).