In molecular biology laboratories, the most frequently studied macromolecules are different types of DNA, RNA and protein molecules. The specific goals of working with them and the techniques required to achieve these goals can greatly vary, although there are a few basic rules which should be kept in case of performing experiments with these types of materials. However, some of these have been already mentioned in the previous chapters, it is important to overview the background of these in this chapter, too.
In general, we want to study protein molecules in a form when they keep their biological activity. For this, their complex higher order structure should be preserved and those effects and reagents, which might destroy these structural features, should be kept away from the solution. For this, we keep the samples on ice during the experiment and protease inhibitors should also be applied. By this, we inhibit reactions that result in spontaneous structure reorganisation and the activation of proteases that might contaminate and degrade our samples. For long term storage, we preserve the samples at -80 °C. However, RNA and DNA samples can be frozen without causing destruction in their structure, proteins might be destroyed by repeated freeze-thaw cycles. Therefore, to store proteins at -80 °C, an antifreeze reagent should be added to the samples (e.g. glycerol) and they should be stored in small aliquots.
Since we generally work with very small quantities of material, we should pay attention for the appropriate handling not to lose them as a result of digestion by contaminating enzymes. Enzymes that degrade
143
DNA, RNA and protein are generally present in our samples since these can be found among enzymes in the cell and are also present on our skin, on laboratory equipment, in the water or on the work bench. To prevent their activity, we frequently use inhibitors of proteases and chelators (e.g. EDTA) that prevent nuclease activity. It is very important to make all precautions to avoid contaminating our samples. For this, use gloves and never touch your pipette tips to avoid the cross-contamination among samples.
Most of the reagents used in molecular biology laboratories are very specific, prepared and purified with great care and consequently are very expensive. Without exaggeration, many of them are more expensive than the same amount of gold. Therefore, be careful, when you use these reagents. You should be aware of the proper quantity of the current reagent you want to use and the proper handling to avoid disruption of the stock solution by contamination and by improper treatment.
Bacterial cell lysis by sonication:
In the laboratory, one of the most frequently used method for bacterial cell lysis is sonication, when cell suspension is exposed to burst of ultrasonic sound. During this technique, cell lysis is caused by cavitation effects (bubbles are formed and collapsed in the liquid due to the ultrasound and the percussion waves raised by the cracking up bubbles generate viscous, dissipative swirls and cell disruption is caused by the shearing stress of them). The sonicator equipment developed for this, excites electrical waves, which is converted to mechanical oscillation,
144
which induces cavitation in an aqueous environment. The device is composed of an oscillator, an amplifier and a wave-converter. In the course of cell disruption by sonication, a lot of heat is generated, therefore the vessel, which contains the cell suspension, should be cooled down by ice or circulating refrigerant. In addition, to prevent overheating of the sample, sonication is most frequently done by alternating cycles of treatment and pauses between them.
Sonication can be used to disrupt cell membranes to release the cellular content. Therefore, sonication is an effective way to release the cellular content but it is not suitable for isolating higher molecular weight DNA, since it results in double stranded breaks. On the other hand, sonication is frequently used to shear chromatin or DNA molecules into smaller fragments, which can be used for chromatin immunoprecipitation experiments or for next generation sequencing.
A sonicator is a relatively simple equipment, since only the intensity, time and frequency should be set on it. The appropriate frequency used for the ultrasonic disruption of bacterial cells is between 15 and 25 kHz.
The user should protect her/his eyes and ears during sonication.
This method has several advantages and disadvantages. The most important advantage is that the treatment can also be used in the liquid-flow procedure. The disadvantages are that it is not appropriate for larger-scale usage, since it is hard to solve the adequate refrigeration and overheating might cause enzyme degradation.
145
Figure 6.1 Sonicator with probe for cell lysis
Figure 6.2 Sonicator with a tank for chromatin fragmentation
146
Practical workflow and protocol
In this practice, we will use the pellet collected from the 5 ml lactose/IPTG induced cell culture.
1. Suspend the cell pellet in 500 ul Buffer I (5 ul protease inhibitor cocktail (PIC) should be added to Buffer I only before usage).
2. Sonicate the samples for 3 x 20 seconds (10 sec break between steps) with 45 % amplitude
2. Centrifuge the samples at 13,000 rpm, 4 °C for 20 min.
3. Transfer the supernatant into a new Eppendorf tube. DO NOT DISCARD THE PRECIPITATE!!
We will also investigate how much protein is present in the precipitate by comparing it to the supernatant samples. Proteins from those cells, which are not lysed, can be found in the pellet therefore we can determine the efficiency of sonication based on this comparison.
4. Suspend the precipitate in 50 ul Buffer I and add 50 ul 2X SDS loading buffer to it.
5. Transfer 20 ul from the supernatant into a new tube and add 20 ul 2X SDS loading buffer to it.
6. Boil these samples for 5 minutes at 100 °C, then keep them at -20 °C until the next practice.
7. Incubate the SUPERNATANT (~450 ul) at 75 °C for 5 minutes, then on ice for 5 minutes.
8. Centrifuge the samples at 13,000 rpm, 4 °C for 20 min
147
Non-thermostable proteins are precipitated and removed therefore in the supernatant only the thermostable proteins (in this case, the Pfu) will be present.
9. Add 20 ul 2X SDS loading to 20 ul SUPERNATANT in a new Eppendorf tube.
10. Boil the samples from step 6. for 5 minutes at 100 °C. Store at -20 °C.
11. Store the residual supernatant at -20 °C to measure enzyme activity later.
148
Figure 6.3 Working procedure during and after sonication
149
Materials
Buffer I:
100 mM Tris pH 7.5
2 mM EDTA 2X SDS loading buffer:
100 mM Tris pH 6.8
200 mM DTT
4 % SDS
0.2 % bromophenol blue
20 % glycerol
150
Short summary (2-3 sentences about the experimental setup):
_________________________________________________________
_________________________________________________________
_________________________________________________________
_________________________________________________________
Used materials (We should check all the solutions and materials before we start the experiment. We should know for what and why we use them during the experimental process.):
151
Changes in the protocol: (the experiment could be repeated only, if we write down everything):
152
Questions
What kind of cell lysis method did we use in this practice?
_______________________________________________________
How does this method work?
_______________________________________________________
_______________________________________________________
Why should you generally keep your samples on ice (or in ice supplemented water bath) during this method? Why isn’t it so important in our case?
_______________________________________________________
_______________________________________________________
_______________________________________________________
What is the function of bromophenol blue in the loading buffer?
_______________________________________________________
_______________________________________________________
What is the function of glycerol in the loading buffer?
_______________________________________________________
_______________________________________________________
153