*3.7.3 Testing for solubility*


<sup>6</sup> In order to have equal loading on an SDS-PAGE gel, the same amount of cells need to be harvested for gel analysis. To harvest the same number of cells each time, calculate the volume in mL needed of your culture that would be the equivalent of 1 mL of OD600 = 0.8. E.g. X mL = 0.8/OD600 of your culture.

#### **Figure 2.**

*SDS-PAGE gel of pre- and post-induction of an RNA binding protein (RBP) in both rich (LB) and minimal (M9) media. The arrow indicates the recombinant RBP.*


### **3.8 Lysing cells**

Traditionally cell lysis can be done with physical disruption or reagent-based methods. Freeze-thaw protocol works best for small volumes (less than 1 mL) in 1.5 mL microcentrifuge tubes. Sonication can be done with smaller volumes using a microtip.

#### *3.8.1 Freeze-thaw*


#### *3.8.2 Sonication*


**49**

(Buffer C).

samples.

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

bursts of 15 s followed by intervals of 30 s for cooling).

3.Load the entire volume of sample onto a 4–12% Bis-Tris mini gel.

5.At the end of the electrophoresis, wash the gel in deionized water three times.

The solubility of a protein depends strongly on the composition of the lysis buffer. Using the procedure described below, the solubility of a specific protein can be tested under neutral (Buffer A), high salt (Buffer B), and with detergent included

1.Follow Section 3.7.1 for the best expressing condition and collect four induced

3.To each cell pellet, add 100 μL of the appropriate buffer (see Section 2.10).

2.Spin down the cells for 5 min at 12,000× g in a microcentrifuge.

5.Lyse cells using the freeze-thaw method (Section 3.8.1).

6.To the supernatant, add 25 μL of 4X SDS-PAGE loading buffer.

7.To the cell pellet, add 125 μL of 1X SDS-PAGE loading buffer.

9.Vortex briefly and then centrifuge for 5 min at maximum speed.

10.Load 20 μL on an SDS-PAGE gel; avoid disturbing the pellet.

7.Wash the gel with deionized water extensively until the water is clear.

3.Insert clean tip of a sonicator in the sample without contacting sides or bottom

4.Set the output power, cycle, and timer to the optimal settings (e.g., five short

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

5.Keep the suspension at all times on ice.

2.Heat the sample at 70°C for 10 min.

4.Run the gel at 200 V for 35 min.

**3.10 Testing lysis conditions for solubility**

4.Vortex to resuspend the cells.

8.Heat all samples to 95°C for 5 min.

1.Add ~100 μg of protein to SDS sample buffer.

6.Stain the gel with Coomassie Blue stain for 1 h.

of the tube.

**3.9 Gel electrophoresis**

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


#### **3.9 Gel electrophoresis**

*Growing and Handling of Bacterial Cultures*

7.Centrifuge for 5 min at 16,000× g at room temperature.

*(M9) media. The arrow indicates the recombinant RBP.*

fuge tube) in a − 80°C freezer, leave for 15 min.

securely in the ice-saltwater bath.

8.Analyze 15 μL of each sample using SDS-PAGE following Section 3.9.

Traditionally cell lysis can be done with physical disruption or reagent-based methods. Freeze-thaw protocol works best for small volumes (less than 1 mL) in 1.5 mL microcentrifuge tubes. Sonication can be done with smaller volumes using a

*SDS-PAGE gel of pre- and post-induction of an RNA binding protein (RBP) in both rich (LB) and minimal* 

1.Freeze the samples to be lysed (typically 0.1–1.0 mL in a 1.5 mL microcentri-

2.Thaw immediately in a 42°C water bath. Vortex vigorously to mix well.

4.Spin the tubes for 5 min at maximum speed in a microcentrifuge.

insoluble protein) by pipetting out the supernatant to a clean tube.

3.Repeat the two previous steps three more times (four freeze-thaw-vortex

5.Separate the supernatant (contains soluble protein) from the pellet (contains

1.Prepare ice-saltwater bath by sprinkling salt over packed ice in a container.

2.Place a 50-mL conical tube containing the cell pellet suspended in lysis buffer

**48**

**3.8 Lysing cells**

*3.8.1 Freeze-thaw*

cycles in all).

*3.8.2 Sonication*

microtip.

**Figure 2.**


#### **3.10 Testing lysis conditions for solubility**

The solubility of a protein depends strongly on the composition of the lysis buffer. Using the procedure described below, the solubility of a specific protein can be tested under neutral (Buffer A), high salt (Buffer B), and with detergent included (Buffer C).

