Hello everyone. It is the 8th week of SIP/MP and its my turn to blog again. This week I will blog on Chloroform Shock its relevance to my project.
Chloroform Shock is a method which is commonly used in the extraction of periplasmic proteins of gram-negative bacteria. Gram-negative bacteria is not only enclosed by its cell membrane, but also a periplasm. Many periplasmic proteins can be found within the periplasmic space (the region between the inner and outer membrane) of the bacteria. Hence, to extract these periplasmic proteins, chloroform is used.
Chloroform is a solvent which is relatively unreactive, miscible and volatile. It has to be said the exact mechanism of action of chloroform shock is currently not known. However, scientiests have been using this technique to extract periplasmic proteins. It is postulated that chloroform form pores in the outer cell wall of the gram-negative bacteria and releasing periplasmic proteins out into the extracellular environment. The optimal time for chloroform to work is 15 minutes for extract of periplasmic proteins only. If the time taken is too short, it may not have reached/penetrate the periplasmic space and periplasmic proteins are not extracted. When the time taken is too long, it may penetrate the periplasmic space and will extract other intracellular proteins as well, such as cell membrane proteins which is irrelevant to my project. Thus, great care must be taken to ensure reaction of the bacteria (cell pellet) with chloroform is 15 minutes exactly.
The use of chloroform is not without its risks. The bottle of chloroform is always opened within the Biosafety Level II Cabinet (which i am using) and not outside it. This is because accidental inhalation can be bad for the health. The role of the Biosafety Cabinet level II is to protect the user as well as the speciman. In Class II cabinets, there is always stream of inward air moving into the cabinet. This is called the inflow and prevents the aerosol generated during any microbial work, to escape out of the Cabinet (air can only flow into the BSC but not out). The inflow can only reach the front inlet grill, just in front of the operator. This is to ensure that unfiltered air outside the BSC cannot enter the BSC and thus there will not be any contamination. A special feature of BSC Level II cabinet is the HEPA-filtered air stream which causes air stream to flow downwards inside the BSC after sucking air from above and filtered. This flushing is called downflow and protects samples within BSC from contamination. In the case where chloroform is accidentally inhaled or consumed, it can depress the nervous system and cause dizziness, fatigue and headache. Hence the appropriate personal protective equipment to wear is a pair of gloves, labcoat and work inside the Biosafety Level II cabinet when performing chloroform shock.
Basic Outline of Chloroform Shock (sorry i cannot give the full details such as centrifuge speed, volume, etc as my supervisor do not allow, hence here is only the basic workflow)
1. Centrifuge to obtain cell pellet (from broth culture)
2. Wash cell pellet
3. Resuspend with PBS (phosphate buffer saline)
4. Repeat step 1 and 2 three times
5. Take OD600 reading
6. Calculate volume of cells to dispense into 5 eppendorf tubes
7. Centrifuge the 5 eppendorf tube
8. Decant supernatant and mix gently
9. Add chloroform and incubate 15 minutes
10. Add Tris-HCl
11. Centrifuge the eppendorf tubes
12. Extract the periplasmic proteins from the supernatant
Thanks for taking your time to read my entry and have an enjoyable and fruitful SIP for the next 12 weeks.
From: Ma Xianwei Benjamin