(A) Protein expression
(i) Test Expression Protocol
Grow small culture (5-50mL) to OD600 0.6 to 0.8
- Remove 1mL to eppendorf tube, pellet bacteria and discard sup. Save pellet for later analysis.
- Induce the rest of the culture with IPTG. This can be split between different temperatures or [IPTG] if desired
- After induction remove 1mL of the induced culture and pellet the bacteria, discard the sup.
- Resuspend the pre/post induction pellets in 200uL PBS.
- Sonicate each sample briefly with the small tip until clear (30 seconds or so)
- Spin each sample in the microfuge for 10 minutes at 14, 000 RPM
- Remove the sup to a new tube.
- Resuspend the pellet in 50uL of 1X sample buffer
- Run 5uL of the sup on SDS-PAGE along with 5uL of the pellet fraction for both pre and post induced samples after boiling for 5 minutes.
- Look for presence of induced band in sup or pellet fractions
(ii) Expression conditions:
Each protein differ in their expression conditions. The ideal conditions should give the least amount of inclusion bodies and highest amount of soluble proteins.
Safest condition: Grow to mid log (around 0.5-6) at 37C, add IPTG, cool to 20C, grow over night. Less inclusion bodies but less soluble proteins as well because pTac is not very effective at this temperature. The tac promoter is most active around 30C to 37C. It is not active at temperatures less than 25C. At cooler temperatures, E. coli will activate cold shock responsive promotors and shut down tac.
Other conditions to try:
- Grow to mid log at 0.4 (or any point between 0.4-0.8) at 37C, add IPTG, grow for 1-4 generations more (by measuring the OD) at 37C.
- Grow to mid log at 0.4 at 37C, add IPTG, cool to 30C, grow for 2-4 generations at 30 C.
- Grow to mid log at0.4 at 37C, add IPTG, cool to 25C, grow for 2-4 generations at 25 C.
François Baneyx lab at UW chemical engineering has done extensive work on cold shock promoters. These promoter systems are available as pCOLD vectors from Takara/Clonetech. According to Baneyx lab, pTac is not active at cold temperatures less than 20C. In these temperatures, only cold shock proteins are produced under the regulator CspA.
If you cannot use E. coli to express proteins, a second option is the yeast pichia_system. It is easier than insect cells.
(B) Bradford Assay (for our DU640 spectrophotometer)
Used to determine the concentration of the protein in the fraction
Pipette 1mL of Coomasie Plus Protein Assay Reagent into curvettes
Prepare standards by adding BSA to six curvettes ( 0, 2, 4, 6, 8, & 10uL). Mix with parafilm.
Two curvettes will be for the sample, add small amounts (5uL, 2uL) so that they appear to be within range of the standards.
Spec: vis on protein app measure the standards first then the samples (dilution correction off)
Use the measurements to determine the concentration based on the volume of sample added to the curvettes.
…More on Bradford:
Frequently, for curve fitting, linear regression is used, even if the standard curve does not look linear. The following example compare linear versus polynomial regression for curve fitting for the same protein. It also shows how the kind of standard (BSA versus gamma globulin) affect the concentration measurement.
|2.5ul BSA in 1ml Bradford||5.04||4.81|
|5ul BSA in 1ml Bradford||11.21||10.83|
|10 ul BSA in 1ml Bradford||18.40||19.29|
|2.5ul B-gg in 1ml Bradford||4.65||5.03|
|5ul B-gg in 1ml Bradford||13.70||12.92|
|10 ul B-gg in 1ml Bradford||24.26||23.66|
(C) Protein Storage.
A cryoprotectant should be added just before flash freezing in liquid Nitrogen, such as Glycerol in 20-50%. Add a reducing agent (preferably just after eluting the protein), and reducing agent should be continuously present during the life time of the protein, such as in dialysis buffers, during freezing, during assays. beta-ME can be added during Nickle affinity purification. We keep it at 5mM, though up to 15mM has been reported as safe. Higher amounts will reduce the Nickle (which will become brown). Use TCEP if you are performing ITC experiments. DTT and beta-ME are not very compatible with ITC. If your protein has a tendency to precipitate, dilute it to less than 1-0.5mg/ml just after elution. Glycerol is not a magik chemical, as people wisely think. If the protein is going to aggregate, it will. Try adding potassium (up to 100mM) or Arginine (100-500mM) and other stabilizers. You can also run the HTS protocol listed in section F (High throughput protocol for Protein Refolding) to find the best stabilizer and use that as a cryostorage, in addtion to 10% glycerol. Add an antimicrobial that won’t affect the downstream application if the protein is used at room temperature for prolonged period. I prefer Triclosan (after testing these won’t affect the downstream application).
(D) Stripping and cleaning GE Hi Trap columns-
- Wash the column with 5 column volumes (CV) of 0.1 M EDTA (Catalog Number ED4S), pH 7.0–8.0.
- Wash the column with 2 CV of deionized water.
- Wash the column with 5 CV of 6 M guanidine HCl, pH 7.5.
- Wash the column with 5 CV of deionized water.
- If using immediately,
- charge the column with 5CV of 100mM Nickle Sulfate.
- Wash with 5CV of deionized water.
- Equilibrate with protein purification buffer, such as TBSG, 5CV.
- Load the protein.
- Load into AKTA.
- If not using immediately, then re-equilibrated with 2 column volumes of 30% ethanol and store at 4C
- Note: The affinity gel can also be cleaned 1 % SDS Wash the column with 5 column volumes of deionized water, then cleaned with a gradual concentration gradient of ethanol. This can be done after the water wash subsequent to the Gua-HCL step . But the concentration percentage of EtOH must be gradually increased and decreased in increments of no more than 25% (i.e., 25, 50, 75, 100, 75, 50, 25, 0) to prevent rapid volume changes of the affinity gel.
(E) 2D Gel Protocol (adapted from Alex Sherl-)
This is a long protocol. So here is the Link to the word file