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Methods in Biochemistry - Coggle Diagram

- - - - Plasmid is given to the bacterial cell to produce a specific protein
        
        Grow a bacterial culture
        
        The bacterial cells are lysed to release "cell stuff"
        
        Centrifuge the mixture to separate from the cell debris
        
        From here, the target protein must be further separated from other soluble proteins (purify with chromatography)
        
        Confirm with gel electrophoresis
  - - - Large porous beads are loaded. The small (target) protein is caught in the beads and takes longer, but the larger protein passes through
    - - Beads in the loading have specific charges designed to attract a certain protein. For example, the beads are negative and the target protein is positive, so it is slowed down while other negative proteins pass through.
        
        A high salt solute is used to elute the target protein
    - - Beads are loaded with a specific chemical attached (example: glucose) to catch the target protein and slow it down
        
        The protein is eluted by passing a high concentration of whatever chemical is used (example glucose) through to release the target protein
    - - Since histidine has a high affinity for nickel, beads bound to nickel are loaded. Synthetically produced proteins can have a poly His-tag attached to their amino acid sequence to grab onto the nickel
        
        For elution, imidazole (which looks similar to histidine), floods the area and competes for binding
  - - - These beads bind to the target protein and the rest of the proteins flow off (separation)
        
        Removal of the target protein (elution)
- - - - Sodium dodecyl sulfate (SDS) is a detergent used to denature proteins
        
        SDS is amphipathic (hydrophilic and hydrophobic regions)
        
        1 molecule of SDS binds for every two amino acids (same charge-to-mass ratio) and coats the protein after it is denatured
      - Beta-ME is used to break disulfide bonds
      - Proteins are then boiled for five minutes just to ensure they are denatured completely
    - - Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to denature and separate the proteins
  - - - Acidic proteins have lots of negatively charged amino acids and will have a pI of less than 7
      - Basic proteins will have lots of positively charged amino acids and will have a pI of greater than 7
- - - - In an antigen coated well, primary antibodies bind to the antigen it is attracted to. The secondary antibody binds to the primary antibody, and an enzyme is linked to this secondary antibody. A chemical reaction will occur when substrate is added, and the extent of color formation is correlation to the amount of specific antibody present.
      - A 96 well plate is used in ELISA
    - - Proteins are separated with SDS-PAGE, transferred to a sheet of polymer (which has high affinity to proteins), and stained with a fluorescent antibody
      - Primary antibody binds to antigen and secondary antibody binds to primary antibody and has something that will produce fluorescence
  - - - These sites have complementary shapes and high-binding affinity