Week 12 Attachment Sharing

Hey guys! 12 weeks have passed. I have started with a second project that covers expression proteomics of a clinical isolate of Stenotrophomonas maltophilia, a nosocomial infectious agent. Expression proteomics relates to studying the entire secretory profile of the bacteria. In my case, i will be precipitating the secretory proteins from S. maltophilia grown at both 28˚C and 37˚C.

Methods
1) Isolate is streaked on an LB agar plate and incubated at 37˚C for 24 hours
2) A colony will be inoculated into 20mL of LB broth and incubated at 28˚C for 24 hours
3) 5x10^7 cells will be inoculated into 12 tubes in which 6 will be incubated at 28˚C and the
other 6 will be incubated at 37˚C
4) Secretory proteins in the supernatant after centrifugation will be transfered to teflon tubes
5) Equal volumes of 40% TCA in Acetone as the supernatant will be added to precipitate the
proteins
6) Tubes will be incubated at 4˚C for 1 hour and inverted every 10 mins
7) Tubes are then centrifuged at 16,000xg for 1 hour at 4˚C
8) Keeping an eye on the protein pellet, supernatant is decanted
9)Remaining supernatant is aspirated out and 250uL of pre-chilled Acetone is added
10) Acetone is used to wash the proteins
11) Washed protein is transfered to a fresh Eppendorf tube and centrifuged
12) Supernatant is aspirated and pellet is allowed to air-dry
13) 200uL of Rehydration Buffer is used to rehydrate the protein pellet
14) 2uL of Protease inhibitor was added to prevent denaturation of proteins
15) Bradford Assay is performed to determine the final protein concentration
16) If there is sufficient protein sample, a 2-Dimensional gel can be run
17) Spots containing significant proteins can be identified and excised
18) Protein spots are then analyzed through the MALDI TOF/TOF
19) Peptides can then b identified through the Mascot Search program

Chemicals
That was a brief itinery of what is carried out in my experiments daily. A single experiment can last for up to 2 weeks. Now i'll explain about certain chemicals that are required for protein precipitation.

Acetone is used for the washing of protein pellets after precipitating with 40% TCA in Acetone. Acetone is the simplest and most important of the ketones. It is a polar organic solvent and therefore dissolves a wide variety of substances. It has low chemical reactivity. These traits, and its relatively low cost, make it the solvent of choice for many processes. About 25% of the acetone produced is used directly as a solvent. Acetone is also used as a drying agent, due to the readiness with which it binds to water, and its volatility. Also, by washing with Acetone, any impurities that have accumulated in the process is removed from the proteins.

Since Acetone can act as a drying agent, the protein pellet will be rid of most liquids thus Rehydration Buffer is required to rehydrate the proteins. The Protease Inhibitor is used to prevent any proteases from acting on the precipitated proteins. Proteases may still be present because they ar enzymes which are also proteins.

Aseptic techniques
When working with proteins to perform analysis, it is important to not come into contact with the samples since it can contribute to the quality and quantity of proteins analyzed. For example, if we do not wear gloves and come into contact with the sample, keratin may be found abundantly in our protein sample. If we talk into our tubes during air-drying, enzymes may be added to our samples.

Conclusion
Thus, i conclude by saying that even though research may seem easy to some and boring to others, i have to say that they are very wrong. I have enjoyed my 12 weeks at the research centre so far through all the ups and downs and all the "failed" experiments. It is inevitable not to get good results but they ARE results so we learn from them. This is the end of my second entry after 12 weeks. Hope to blog in 6 weeks time with more interesting facts to share. See you guys at the next Campus Discussion!

Johanna
0503309G
TG02