L2: Protein Isolation & Protein Assay & SDS-PAGE

What are enzymes?

• biological catalysts (proteins) that speed up chemical reactions in living organisms without being consumed in the process.

When a cell is lysed to extract protein out, what is in the supernatant layer and what is in the pellet form?

Supernatant:

• protein

• DNA/RNA

• Salts

• Sugars

Pellets:

• lipids

• Bulky complex carbs

What should you AVOID to protect proteins from being damaged?

• Heat

• Strong Acids

• Strong Bases

What are good techniques to use to create a lysate?

1. Mechanical

2. Chemical detergents

3. Grinding: mortar & pestle

4. Sonicators

What is the procedure of extracting proteins from the tissue?

1. Tissue is lysed: to break open the cell to extract the proteins out

2. Centrifuge: to pellet the debris

3. Remove supernatant: proteins and others are in the supernatant while the debris is in the pellet form

4. Add 10% TCA (Trichloroacetic Acid): to precipitate the protein and pellet into solid form allowing protein and water to separate

5. Centrifuge: to pellet the proteins

6. Remove supernatant

7. Wash with alcohol & allow to dry

8. Add water: to get protein in soluble liquid form

What are the cons of performing a lysate on tissues to extract protein?

It can ruin the SHAPE of proteins

• can prevent you from using the Native Protein Isolation Strategy

What are 2 isolation strategies of protein?

1. Native proteins: folded naturally

2. Denatured proteins: protein unfolding down to primary structure

What technique is used to quantify protein?

Protein Assay

What is protein assay technique?

Method used to measure the concentration of protein in a sample

What are the 3 types of protein assay techniques?

1. Bradford assay

2. Lowery assay

3. BCA assay

What is a Bradford Assay

• uses Coomassie Blue Dye, which binds to protein

• When the dye reacts with protein it changes color from brown to blue

• Quick and easy

What is a Lowry Assay

• interacts with Copper Ions to make a blue color when it reacts with protein

What is BCA Assay?

• Reacts with Copper and changes color to purple when it reacts with protein

What happens when there is too much protein in an SDS-PAGE?

Data will be too difficult to analyze

What happens if there is not enough protein in an SDS-PAGE gel?

The bands will be too faint to analyze in the data

What technique is used to analyze the concentration of protein?

Spectrophotometer

• measures the absorbance of LIGHT at specific wavelengths

What is an SDS-PAGE?

Sodium Dodecyl Sulfate (SDS) is a detergent

• it is a Protein Gel that separated proteins based on Molecular Weight

Why are proteins separated based on molecular weight?

Some proteins can be the same size but with different weight due to amino acids differing by their R-groups

What is the PAGE mean in SDS-PAGE?

Polyacrylamide Gel Electrophoresis (PAGE)

• sticks together by cross-linking fibers via chemical reactions

What type of technique is PAGE?

• Used to separate proteins or nucleic acids based on their SIZE and CHARGE.

• The molecules are loaded into a Polyacrylamide gel and moves through it when an electric current is applied

What are the 4 forces that help hold protein in their tertiary shape?

1. Hydrogen bonds

2. Ionic bonds

3. Hydrophobic interactions

4. Disulfide bonds

What are hydrogen bonds?

• breaks down by using HEAT

• Forms between polar side chains or backbone groups

What are ionic bonds?

• reducing agent: SDS

• Occurs between oppositely charged side chains

What are hydrophobic interactions?

• reducing agent: SDS

• Nonpolar side chains cluster away from water in protein

What are disulfide bonds?

• Reducing agent: DDT or Beta-ME

• Covalent bonds that give a strong structural support

Disulfide bridges can be broken down by using what reducing agents?

DDT or Beta-ME

In an SDS-PAGE gel, what type of liquid is used to place the gel in?

Electrolyte buffer

What are the units for the size standard in an SDS-PAGE gel?

KiloDaltons (KDa)

What is in a protein sample load for an SDS-PAGE

1. DDT

2. SDS

3. HEAT

4. Loading Dye

In an SDS-PAGE, what are the 2 types of dyes used to stain the gel after running to visualize proteins?

1. Coomassie Blue

2. Silver Nitrate

Proteins can be a mixed charge of positive charges or negative charges, what can be used to neutralize the charge?

SDS can be used as a detergent