Team:UCLA/Notebook/Protein Cages/21 August 2015
Intro: Grew the starter culture overnight for ~16 hours. Refreshed into 300 mL expression culture. Induced with IPTG.
Expression Culture:
OD with 4 mL of starter culture: 0.023
OD with all of starter culture: 0.049
Incubation with all of starter culture began at 1:25 pm.
OD after one hour: 0.073
OD after two hours: 0.112
OD after 3.5 hours: 0.443
OD after 4.5 hours: 1.091
Induced with IPTG to 1mM concentration at ~6:20pm.
OD with IPTG after one hour: 1.370
OD with IPTG after two hours: 1.390
Cells pelleted after three hours of induction at max speed for 20 min. Washed with sterile ddH2O. Repelleted. Frozen in -20C
Tyler Lee --Wtleeiv 01:24, 22 August 2015 (CDT)
Phillip's notes:
Introduction: Lysis and purification of PCquad Y will be done today.
Procedures:
-Lysis protocol:
1. Obtain the frozen cell pellet, keep on ice. Cell pellet was 0.095
2. To the frozen cell pellet, add lysis buffer to a ratio of 1:10 grams of cell pellet to mL of lysis buffer.
3. Incubate 5 minutes on ice.
4. Vortex vigorously for 20 seconds, incubate on ice for 5 minutes. A pipette can be used to carefully re-suspend the pellet if vortexing is insufficient. At this point, the DNA seemed to precipitate and the solution was very viscous. 2uL of DNase was added and the tube was incubated for 20-25minutes until it was clear.
5. Repeat Step 4 two to four times, until the pellet is sufficiently broken up.
6. Centrifuge at 4C at max speed for 10 minutes. Retrieve supernatant (lysate). The pellet can be kept for downstream analysis if desired.
7. Store at -80C or -20C (-80 recommended) until use.
Ni-NTA Affinity purification (modified from the Thermo protocol):
Buffers:
1. Equilibration buffer: a. 50mM phosphate buffer (pH 8) b. 300mM NaCl c. 10mM imidazole (pH 7.4) 2. Wash buffer: a. 50mM phosphate buffer b. 300mM NaCl c. 25mM imidazole 3. Elution buffer: a. 50mM phosphate buffer b. 300mM NaCl c. 250mM imidazole
-Procedure
1. Add one volume of resuspended Ni-resin to a container with 3-4 times the volume of resin added. 300uL of resin was used.
2. Add two resin bed volumes of equilibration buffer, mix by rotating for 5 minutes. Ensure that the resin is fully resuspended at each step.
3. Centrifuge for 2 minutes at 700g. Discard supernatant.
4. If the above lysis buffer was not used, add protein sample to equilibration buffer at a 1:2 v/v ratio for a total volume greater than the resin bed volume if (Usually two volumes). For example, for 1mL of resin, add (1mL of protein sample + 1mL of equilibration buffer). Save at least 20uL of whole cell lysate for SDS-PAGE analysis.
5. Add protein to resin, rotate for 30 minutes. Keep cold if possible.
6. Centrifuge at 700g for 2 minutes. Remove supernatant, save for analysis.
7. Add two resin-bed volumes of wash buffer. Rotate for 5 minutes. Centrifuge. Remove supernatant. Save for analysis.
8. Repeat wash 3 to 5 times (more if necessary).
9. Elute in one resin-bed volume of Elution buffer. Rotate for 10 minutes. Centrifuge 2 min at 700g. Save supernatant for analysis.
10. Repeated elution 3 times. A fourth elution volume was added, and the tube was left on the shaker over the weekend to see what happens.
Conclusions: DLS will be ran next week, and SDS-PAGE will be ran when gels come in.