**2.12 Uniform 15N/13C labeling of recombinant proteins**

1.LB.

2.IPTG.


6. 10X Ammonium chloride: 93.5 mM NH4Cl.


## **2.13 Protein purification using immobilized metal affinity chromotography (IMAC)**

Immobilized metal affinity chromatography (IMAC) is a common method for affinity purification. A genetically encoded 6-histidine repeat affinity tag can be introduced to the carboxy or amino terminal end of the protein during cloning, which has high affinity for metal ions. The protocol given here is for affinity purification by immobilization of nickel ions with a chelator molecule, nitrilotriacetic acid (NTA) that is covalently bound to agarose; commonly known as Ni-NTA agarose. The following buffers are meant to represent a general starting point. Depending on the pI of your recombinant protein and the propensity to nonspecifically interact with the column material or resident *E. coli* proteins, modifications may need to be made. Additional purification may be necessary, especially when purifying proteins that bind to nucleic acids. A lithium wash may be added to the Ni-NTA purification

**43**

*Growing and Handling of Bacterial Cultures within a Shared Core Facility for Integrated…*

phy are often added in addition to a nickel affinity purification step.

1.Lysis buffer: 0.1 M Tris-HCl, 0.1 M NaCl, pH 8.1.

2.Wash buffer: Lysis buffer plus 5–20 mM imidazole.

3.Elution buffer: Lysis buffer plus 100–300 mM imidazole.

**2.14 Differential scanning fluorimetry to assess protein stability**

2.qPCR machine with filter set that matches fluorescent dye and equipped with a

1.Inoculate 5 mL of LB (or SOB if preparing DH5α cells) with 10 μL of appro-

2.Use the overnight culture to inoculate 250 mL of LB (or SOB if preparing

DH5α cells) and incubate at 30°C until the optical density at 600 nm (OD600) is

3.Chill the culture for at least 10 min on ice. For steps 4–10, keep the cell suspen-

5.Gently resuspend the pellet in 50 mL ice-cold CC buffer into 50-mL conical tubes. Resuspend with a 10-mL serological pipette and avoid introducing

<sup>2</sup> Some cell lines have a resident plasmid, such as BL21(DE3) pLysS or pLysE cells and require addition of

and grow overnight at 37°C and 250 rpm in a shaking

1.Low ionic strength buffer (e.g., 10 mM Tris-HCl).

4.96-well polymerase chain reaction (PCR) microplate.

to remove nucleic acids. Ion exchange, heparin affinity, size exclusion chromatogra-

*DOI: http://dx.doi.org/10.5772/intechopen.81932*

4.Probe sonicator.

7.AEBSF.

**3. Methods**

priate *E. coli* cells2

between 0.4–0.6.

incubator.

sion on ice.

bubbles.

5. 1 mg/mL lysozyme.

6.Protease inhibitor cocktail.

ramp rate of minimum 1°C/min.

3.A fluorescent dye that will bind proteins.

**3.1 Preparation of chemically competent cells**

4.Spin the cell suspension for 10 min at 6000× g.

antibiotics for selection of cells containing those plasmids.

<sup>1</sup> The stock solution of 10 mg/mL is above the solubility limit of biotin, do not sterile filter this solution. Simply make the solution with previously sterilized water.

*Growing and Handling of Bacterial Cultures within a Shared Core Facility for Integrated… DOI: http://dx.doi.org/10.5772/intechopen.81932*

to remove nucleic acids. Ion exchange, heparin affinity, size exclusion chromatography are often added in addition to a nickel affinity purification step.


*Growing and Handling of Bacterial Cultures*

3.Culture tubes and flasks.

3.Culture tubes and flasks.

7. 20% wt/vol glucose stock.

4.Incubator/shaker.

8. 100 mM CaCl2.

9. 1.0 M MgSO4.

10.10 mg/mL thiamine.

12.Antibiotic for plasmid selection.

Simply make the solution with previously sterilized water.

11.10 mg/mL biotin.1

**(IMAC)**

4.Incubator/shaker.

1.LB.

1.LB.

2.IPTG.

2.IPTG.

**2.11 Large-scale expression of recombinant proteins**

**2.12 Uniform 15N/13C labeling of recombinant proteins**

6. 10X Ammonium chloride: 93.5 mM NH4Cl.

5. 10X M9 medium: 340 mM Na2HPO4, 220 mM KH2PO4, 85.5 mM NaCl, pH 7.4.

**2.13 Protein purification using immobilized metal affinity chromotography** 

Immobilized metal affinity chromatography (IMAC) is a common method for affinity purification. A genetically encoded 6-histidine repeat affinity tag can be introduced to the carboxy or amino terminal end of the protein during cloning, which has high affinity for metal ions. The protocol given here is for affinity purification by immobilization of nickel ions with a chelator molecule, nitrilotriacetic acid (NTA) that is covalently bound to agarose; commonly known as Ni-NTA agarose. The following buffers are meant to represent a general starting point. Depending on the pI of your recombinant protein and the propensity to nonspecifically interact with the column material or resident *E. coli* proteins, modifications may need to be made. Additional purification may be necessary, especially when purifying proteins that bind to nucleic acids. A lithium wash may be added to the Ni-NTA purification

<sup>1</sup> The stock solution of 10 mg/mL is above the solubility limit of biotin, do not sterile filter this solution.

**42**
