Proteomics

Proteome :  proteome" is a mix  of "protein" and "genome While genome is the entire heredity information of an organism , proteome is the organism's complete complement of proteins. Proteomic : Is the   study of an organism's complete complement of proteins especially their function and structure . While genomic is the study of the whole genome.

Why not mRNA : Proteomic consider the amount and physiology of the protein in the cell mRNA is not always translated to protein . A lot of proteins are modified after the RNA been translated.

Protein Structure Protein Biochemistry NMR: Nuclear magnetic resonance spectroscopy X-ray crystallography Structural Biologists 10’s of mg quantities Protein Biochemistry Westerns pull-down assays Enzyme Assays Sub-mg quantities مطياف الرنين المغناطيسي النووي، والأكثر شيوعا المعروفة باسم مطياف الرنين المغناطيسي النووي، هو أسلوب البحوث التي يستغل الخواص المغناطيسية للالأنوية الذرية معينة لتحديد الخصائص الفيزيائية والكيميائية للذرات أو الجزيئات التي ترد فيها. فإنه يعتمد على ظاهرة الرنين المغناطيسي النووي ويمكن أن توفر معلومات مفصلة عن الهيكل، وديناميات، رد فعل الدولة، والبيئة الكيميائية للجزيئات

Western Blot : 1- Making the SDS gel

SDS-PAGE Gel Component Table

Finally the cascade system is ready

2- transfer the proteins to nitrocellulose membrane : By Trans-Blot that is specifically designed to pass electric current horizontally through the gel forcing the negatively charged proteins to migrate out of the gel onto the nitrocellulose membrane.

3- Primary antibody is added to the membrane and incubated to allow the antibody to bind to the myosin protein on the membrane. The unbound antibody is then washed away.

4- Secondary antibody is added to the membrane and incubated to allow the secondary antibody to bind to the primary antibody. The unbound secondary antibody is then washed away.

5-Colorimetric enzyme substrate is added to the membrane and incubated to allow color to develop. Purple/gray bands will develop on the membrane exactly where the myosin protein bands are located.

1. If not blocked overnight, immerse membrane in 25 ml blocking solution for 15 minutes to 2 hours at room temperature on a rocking platform. 2. Discard blocking solution and incubate membrane with 10 ml of primary antibody for 10–20 minutes on rocking platform set to a faster setting to ensure constant coverage of the membrane. 3. Quickly rinse the membrane in 50 ml of wash buffer then discard the wash. 4. Add 50 ml of wash buffer to membrane for 3 minutes on rocking platform at a medium speed setting. 5. Discard the wash and incubate membrane with 10 ml of secondary antibody for 5–15 minutes on rocking platform set to a fast setting. 6. Quickly rinse the membrane in 50 ml of wash buffer and discard the wash. 7. Add 50 ml of wash buffer and wash membrane for 3 minutes on rocking platform on a medium speed setting. 8. Discard the wash and add 10 ml of HRP color detection reagent. 9. Incubate 10–30 minutes, either with manual shaking or on a rocking platform, and watch the color development. 10. Rinse the membrane twice with distilled water and blot dry with paper towel. 11. Air dry for 30 minutes to 1 hour and then cover in plastic wrap or tape in lab book. 3