Protocols
- Protocols
- Competent Cells Protocol
- hTERT-RPE serum starvation for synchronization
- Immunofluorescence Protocol
- hTERT-RPE serum starvation for synchronization
- Flow Cytometry Manuals
- Fork regression/restoration/footprinting
- SMARCAL1 DNA binding ATPase assays
- PI and BrdU Flow Cytometry Protocols
- Rong’s 293T suspension+mass spec
- Lysis with DNAse for all nuclear proteins including histones
- DNA Combing Protocol
- DNA fiber labeling
- Comet Assay
- Nascent ssDNA IF assay
- 53BP1 foci
- Retro/Lentivirus production
- PEI transfection of 293T cells
- siRNA transfection optimization
- Flag-His purification mammalian cells
- ATRflox cells Cre
- ATR kinase assay
- G2/M checkpoint assay
- Freezing/Thawing cells in Liquid Nitrogen
- RPA-ssDNA pull down
- GST protein purification from bacteria
- Bacterial expression/His purification
- HeLa siRNA/plasmid transfection
- Protein solubility test in E.coli
Testing recombinant protein production and solubility in E.coli.
- Transform BL21 codon plus or equivalent cells with plasmid. Plate on appropriate antibiotic plate. The BL21 codon plus should be plated with choloramphenicol (50ug/ml) to select for the extra tRNAs. Grow overnight.
- Pick 2 transformants into 2ml of 37degree LB/antibiotic media and also streak a portion of the same transformant onto a new LB/antibiotic plate to save. I usually will add chloramphenicol
- Grow until visibly turbid (usually about 5h, O.D. of approximately 0.4-0.6). Remove 60ul, centrifuge, and freeze cell pellet for uninduced sample. Add IPTG to a final concentration of 1mM. Incubate for another 1.5h.
- Remove 30ul of cells, centrifuge, and freeze cell pellet for induced sample.
- Pellet 1.5ml of the remaining culture in a 1.5ml microfuge tube.
- Resuspend the cell pellet in 300ul of PBS.
- Sonicate to lyse cells using microtip sonicator. Keep samples cold on ice.
- Centrifuge lysed cells full speed for 5 minutes at 4 degrees.
- Supernatant = soluble lysate. Pellet = insoluble.
- Mix 25ul of the soluble lysate with 25ul of 2xSB. Add 50ul of 2xSB to the insoluble pellet. Also mix 30ul of 2xSB with each of the cell pellets that you froze in steps 3 and 4.
- Heat samples to 95-100 degrees for 5 min and vortex thoroughly. Centrifuge. Load 15ul of each sample on an SDS page gel. Stain with Coomassie blue.
Optimizing expression and solubility
Expression levels, solubility, and stability can be affected by time, temperature, and E.coli strain. Start by varying the temperature. Test 37, 30, 25, 18 degrees. Note that as the temperature decreases, the length of induction should increase. So a 1h induction at 37 = 2h at 30 = 5h at 25 = 12h at 18 degrees. Protein induction is usually best if the cultures are grown to an O.D. of about 0.5-0.8 prior to induction. You can grow at 37 then switch to the lower temperatures just prior to induction.
Large-scale induction
I typically grow an overnight culture of about 25-50ml in antibiotic media, then dilute to an O.D. of 0.1-0.15. Grow until O.D. of 0.5-0.8. Induce with IPTG at optimal temperature and optimal time. Be sure to save all fractions to test on SDS-PAGE.
Other variables that can be changed to improve protein production: Change the E.coli strain. Rosetta and BL21 have been most useful. Change the IPTG concentration -- reducing it to 0.05-0.1mM or less may help solubility of protein. Change the growth media – try Terrific Broth or other richer medias. E.coli that has been sitting on plates for several days tend to work poorer than fresh transformants.