Protein Sample Preparation

Protein Sample Preparation

1. Why?

   • The study of proteins and their function is central to understanding both cells and organisms

2. Reasons

   • serve as catalysts
   • receptors that convey information
   • serve as intracellular signaling components
   • key components of the machinery
   • serve as structural elements
   • involved in manipulation of DNA and RNA

3. steps

   • cell disruption
   • extraction
   • removal
   • quantification
   • separation

4. Releasation of protein

   • Mechanical discruption
   • Non-mechanilcal disruption

5. Protein solubilization

   • must well solubilized

6. Protein Quantification

   • A total amount of protein or concentration of protein in a sample can be determined by protein assay
   • Method that can be used are Bradford, Lowry, Biuret or BCA Protein Assay
   • protein quantify in terms of molecular weight relative to a hydrogen atom
     • protein migrate according to molecular weights

7. Electrophoresis

   • Migration of charged molecules towards opposite charge

8. SDS-PAGE

   • A form of electrophoresis that treats samples with SDS to denature proteins

9. Markers (ladder)

   • Migration pattern depend on gel type, gel concentration and running buffer