**3.13 Protein purification using IMAC**


**53**

tubes.

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

14.Pool fractions containing pure recombinant protein and dialyze into an

Ni-NTA agarose may be re-used for the same protein multiple times.

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

1.Prepare 1500 μL of 18 μM protein in dilution buffer.

5.Add 80 μL additive to be screened to one of nine vials.

6.Add 80 μL of dilution buffer in the tenth vial as a control.

15.Clean Ni-NTA agarose by washing with 0.5 M NaOH for 30 min. Wash with five CVs sterile deionized water and store in 30% ethanol for long-term storage. The

4.Divide the protein plus dye solution among 10 vials: 140 μL per vial (some

8.Transfer 50 μL of protein plus dye plus additive solution (or control solution)

9.Cover PCR plate with a sheet of optically clear adhesive and seal each well.

10.Spin 96-well PCR plate in a centrifuge equipped with a plate holder at 800× g

11.Place 96-well PCR plate into qPCR machine and run the following program:

• set the following temperatures: an initial 2 min hold at 25°C, increase in

to a final temperature of

H, 15N 2D

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

appropriate buffer.

2.Add 1.5 μL of 5000X dye.

3.Pipette up and down gently to mix.

7.Pipette up and down gently to mix.

for 2 min at room temperature.

95°C (with a 2 min hold).

12.Export data for further analysis.

equipped with fine temperature resolution.

to the 96-well PCR plate in triplicate.8

• select total volume per well 50 μL

• select experiment type melting curve

increments of 0.5–1.0°C and hold each for 30 s,9

13.Data can be plotted as the fluorescence vs. temperature (**Figure 4**).

14.After buffer optimization, structural data can be collected such as a 1

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

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

NMR spectrum (see **Figure 5** for example of spectrum).

stock solution will remain).

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

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

