Copy of Protein Sample Preparation

1. Protein Quantification

1.1. A total amount of protein or concentration of protein in a sample can be determined by protein assay.

1.2. Example of colorimetric protein assay methods that can be used

1.2.1. Bradford

1.2.2. Lowry

1.2.3. Biuret or BCA Protein Assay

2. Migration pattern of ladder

2.1. Dependable on

2.1.1. Gel type

2.1.2. Gel concentration

2.1.3. Running buffer

2.2. Most commercially available protein ladders have coloured reference bands

2.2.1. For convenience and accuracy during molecular weight estimation

3. Molecular weight markers (ladder)

3.1. Protein standards, are made from proteins that have been genetically engineered to be specific molecular weights

3.2. Bound to dye molecules so that they are visible on the gel or western blot.

4. Sample/Loading Buffer contains

4.1. Tris-Ci

4.1.1. Creates stable environment for the proteins.

4.2. Glycerol

4.2.1. Increases the density of the protein sample making it sinked/easier to load in the well.

4.3. SDS

4.3.1. To denature all proteins by breaking down secondary structure of proteins and make them negatively charged

4.4. Bromophenol blue

4.4.1. Used to track the run of protein sample on the gel

4.5. B-Mercaptoethanol

4.5.1. Necessary to break down disulfide bonds in tertiary structure of proteins.

5. Why study protein?

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

5.2. They serve as catalyst that maintain metabolic processes in the cell

5.3. Serve as structural elements

5.4. They are receptors that convey information about extracellular environment to the cell

5.5. Serve as intracellular signaling components

5.6. Key components of the machinery

5.7. Manipulation of DNA and RNA

6. SDS-PAGE

6.1. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis

7. General principal of Protein electrophoresis and SDS-PAGE

7.1. Electrophoresis is the migration of charged molecules in an electric field towards the electrode of the opposite charge.

7.2. SDS-PAGE is a form of electrophoresis that treats samples with SDS to denature protein.

8. Protein sample preparation

8.1. 1. Cell disruption

8.2. 2. Extraction

8.3. 3. Removal of interfering compounds and changes of the physio/chemical properties

8.4. 4. Quantification

8.5. 5. Separation via SDS-PAGE

8.6. Cells need to be disrupted (to break open)