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


*Growing and Handling of Bacterial Cultures*

**3.13 Protein purification using IMAC**

2.Lyse cells (See Section 3.8).

inhibitor cocktail, and 1 mg/mL lysozyme.

temperature for further SDS-PAGE analysis.

followed by two CVs of the buffer.

analysis.

PAGE analysis.

PAGE analysis.

7.Filter the supernatant over a 0.4-micron syringe filter.

10.Harvest the cells by centrifugation at 6000× g for 20–30 min at 4°C. Discard

1.Resuspend cell pellet in ~35 mL of lysis buffer containing AEBSF, a protease

3.Remove 75 μL of lysate and pipette into a 1.5 mL microcentrifuge tube. Centrifuge the 75 μL aliquot for 10 min at 12,000× g at room temperature.

sample buffer. To the remaining pellet, add 100 μL of 1X SDS PAGE.

4.Separate the supernatant into a new vial and treat with 25 μL of 4X SDS PAGE

5.Boil separated and SDS buffer-treated samples for 10 min and store at room

6.Centrifuge the remaining lysate (ca. 35 mL) at 16,000× g at 4°C for 20–30 min.

8.Pre-equilibrate the appropriate amount of Ni-NTA agarose for the amount of protein expressed in desired equilibration buffer; typically, 1–2 mL of settled agarose washed with two column volumes (CVs) of sterile, deionized water

9.Bind the filtered lysate to the Ni-NTA agarose either batch or column loading. For batch loading, combine the filtered lysate and Ni-NTA agarose and gently rock for 30–60 min prior to pouring into the column. For column loading, pack Ni-NTA agarose into the column and pass the filtered lysate through the column. Collect the flow through eluent for SDS-PAGE

10.Wash the column with 15 CVs of cold lysis buffer. Collect wash eluent for SDS-

11.A step gradient consisting of 15 CVs each of 5, 10, and 20 mM imidazole may be used to determine best washing conditions. Collect wash eluents for SDS-

<sup>7</sup> Store lysate cell pellet at −20°C until SDS-PAGE has confirmed that full extraction of desired protein from the pellet is accomplished. Keep all buffers and protein samples at 4°C during purification. Batch vs. column choice will depend on binding properties of the individual protein. The SDS-PAGE of the step gradient imidazole washes will illustrate what is the highest imidazole concentration that can be used as an initial wash to clean off non-binding contaminants. If large amounts of protein remain in the cell pellet, alternative growing methods, such as IPTG concentration adjustment, or alternative purification methods including purification under denaturing conditions, should be considered. Additional purification may be

(**Figure 3**).

12.Wash the column with 20 CVs elution buffer, collecting 1 mL fractions.

13.Evaluate all collected samples by SDS-PAGE (see Section 3.9)7

necessary, such as ion exchange, or heparin binding column chromatography.

supernatant. Store the pellet at −20°C until ready for cell lysis.

**52**

	- select total volume per well 50 μL
	- select experiment type melting curve
	- set the following temperatures: an initial 2 min hold at 25°C, increase in increments of 0.5–1.0°C and hold each for 30 s,9 to a final temperature of 95°C (with a 2 min hold).

<sup>8</sup> Excess solutions are suggested to account for loss due to transfers and sticking to the sidewall of the tubes.

<sup>9</sup> Slower ramp rates will provide better melting temperature resolution, however not all instruments are equipped with fine temperature resolution.

#### **Figure 3.**

*SDS-PAGE gel of a typical Ni-NTA purification of an RBP (arrow indicates the recombinant RBP). (A) Samples appear in the following order: MW markers, Lysate, Supernatant, Pellet, Flowthrough, Wash #1: 50 mM Tris-Cl, 100 mM NaCl, pH 7.7; washes #2–4: 10 mM Imidazole, 50 mM Tris-Cl, 100 mM NaCl, pH 7.7. (B) MW markers, Elutions #1–9: 200 mM Imidazole, 50 mM Tris-Cl, 100 mM NaCl, pH 7.7. Some protein elutes from the column in the wash steps. All fractions are kept and can be pooled after SDS-PAGE analysis.*

#### **Figure 4.**

*DSF analysis of an RBP in buffer (10 mM Tris-Cl) with different additives. (A) A graph of the fluorescence at 602 nm at increasing temperatures for the surveyed additive screen. The inflection point preceding the peak is the melting temperature. (B) A first derivative plot with a four-point smoothing applied helps to visualize the melting temperature, where the peak is the melting temperature. The legend provides a key for both A and B.*

**Figure 5.** *1 H, 15N 2D NMR spectrum of an RBP prepared using the methods described here.*

#### **4. Conclusion**

We have described the workflow for protein expression and purification used in our shared core laboratory. These methods for growing and handling bacterial cultures work well for plasmid amplification, mini-expression screening, optimized larger-scale protein production, protein isolation and purification, and

**55**

**Author details**

and Julia Thom Oxford\*

provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Lisa R. Warner, Olga Mass, Nancy Donnelly Lenn, Briana R. Grantham

Biomolecular Research Center, Boise State University, Boise, Idaho, USA

\*Address all correspondence to: joxford@boisestate.edu

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

characterization of optimized experimental solution buffer conditions. Future methods can be added as needed by the users of the core and the university research

This publication was made possible by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants P20GM109095 and P20GM103408. The authors

wish to acknowledge Jackson Wall for careful reading and suggestions.

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

Authors have no conflict of interest.

community.

**Acknowledgements**

**Conflict of interest**

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

characterization of optimized experimental solution buffer conditions. Future methods can be added as needed by the users of the core and the university research community.
