M3L1 - Protein Purification

Why purify proteins

• Study the folded 3D structures (xray crystallography)

• Identify protein function

  • What the substrates are

  • What binding to substrate does

• Identify proteins amino acid sequence

  • Predict the seuqnece of gene encoding the protein

• Develope antibodies specific to protein

  • Can be used in immunoflourescence microscopy to detect protein location in cell/tissue

7 Steps to isolate protein

1. Identify unique assay/experiment for protein (how to detect it) 

2. Select protein source 

3. Extract proteins from cell 

4. Solubilize protein 

5. stabilize protein 

6. fractionate (seperate) your protein 

7. Evaluate purity

Protein Assay

• Way of detecting protein 

  • Ex. measuring enzymatic activity if it’s an enzyme by looking for release of a product or use of substrate 

  • Ex. Use antibody to moniter the presence/conc of protein 

  • Ex. if binds to unique substrate like RNA or actin, moniter biological activity to see presence 

Protein Source and Extraction

• Should be easily obtained in large amounts 

  • Studying muscle protein? Get it from muscle cells 

  • Studying Hemoglobin? Use RBC 

• Choose a cell low in similar proteins which may co-purify with your target 

• Protein source must be low in proteases that may destroy target

  • You could add protease inhibitors during purificaiton steps

  • You could also express the rpotein in alternative cell types (like mouse protein in bactera) 

  • Ensures you extract the greatest amount of protein 

• Lysing Cells:

  • Chemical lysis

  • physical grinding

  • ultrasonic sonicators 

Protein Solubilization

• Soluble Proteins:

  • Cytosolic proteins

  • secreted proteins

• Insoluble Proteins

  • Transmembrane proteins

  • Membrane-associated Proteins 

• Factors affecting protein solubility:

  • Ph of Solution 

  • Salt conc

  • Presence of detergents (in more insoluble proteins) 

Detergents: 

• Help stabilize molecular interactions in a protein 

• increases solubility of insoluble proteins in solution

Protein Stabilization

• Maintains native structure and prevents degredation

• You should maintain non-covalent interactions stabilizing the folded protein

Parameters: 

• Temp

• Protease inhibitors 

• Ligands

• Salts

• Metal ions

• Concentration of target protein (may aggregate at high conc) 

• pH

Protein Fractionation

• Seperating proteins into different groups 

• Many techniques are present to take advantage of a chemical/physical property 

• Proteins vary in 

  • charge

  • size

  • polarity

  • solubility 

  • shape

Protein Fractionation Techniques

1. Ion Exchange Chromatography (charge)

2. Gel Filteration Chromatography (Size)

3. Adsorption Chromatography (Polarity) 

4. Affinity Chromatography (Specificity in Binding) 

Protein Fractionation: Charge

• Ion exchange chromatography 

• gel electrophoresis 

Protein Fractionation: Size

• Gel electrophoresis

• gel filteration chromatography

• ultracentrifugation 

Protein Fractionation: Polarity

• Adsorption chromatography

• Hydrophobic interaction chromatography 

Protein Fractionation: Specificity in Binding

• Affinity chromatography 

• Seperates proteins from a protein-protein or protein-substrate binding 

Differential Centrifugation

• Commonly begins protein irolation

• Isolates a subset of proteins based on size or subcellular localization 

Steps:

1. Spin tubes at 1000g

2. Produces a pellet at the bottom with nuclei/chloroplasts while fluid has other cellular components 

   1. Split those based on the protein you’re studying

3. Spin supernatant fluid (if that’s where ur protein is) at 10,000g

4. Produces a pellet with mitochondria

   1. Split those based on the protein ur studying 

5. Spin supernatant fluid (if that’s where ur protein is) at 100,000g

6. Produces a pellet with ER, golgi apparatus, lysosomes, and peroxisomes 

   1. Supernatant fluid is just cytosole for cytosolic proteins 

Chromatography

• Commonly second step of protein fractionation 

• (aq) extract is poured down column with matrix to help sort proteins based on different properties 

• If there’s interaction between protein and beads, it wil be slow to move (blue) 

• Lower interactions means faster to move (pink) 

• thus Pink proteins will come out first 

Ion Exchange Chromatography

• Beads in the column are charged, usually positive 

Example:

• Protein extracts are loaded at top (red and blue)

  • Red = +

  • Blue = -

• Red will flow by faster as + repels +

• Blue will be attracted and move slow 

• To elute blue, the column must be washed 

Washing column

• Main basis: Interfere with ionis interactions

• Way 1: Adding salt solution (NaCl)

  • Cl- will disrupt the interaction of blue and the beads by interacting with the beads

• Way 2: Warm wash solution

• Way 3: change in pH of wash solution

Gel Filteration Chromatography

• Seperates protein by size 

• Beads have small holes in them 

• Small proteins get trapped while larger ones flow past 

  • Fraction 2 will have all the larger proteins

• Removing the beads and washing them/spinning on low speed dislodges small proteins from the beads

  • Beads can vary in size depending on protein of interest

  • Bead sizes are defined upon threshold size (the largest protein that could fit)

Affinity Chromatography

• Seperates proteins based on specificity of binding to another molecule

• Beads are covalently attached to antibody 

• The antibody will associate with a single protein (the antigen) 

• target protein will stay in the column due to non-covalent interactions with the antibody

  • The other proteins elute straight in fraction 1

• To remove the target, pH / temp / [salt] can be changed

• Ex. a GTP-binding protein can be isolated by attaching GTP to the beads

SDS-PAGE electrophoresis

• intentionally denatures protein 

• Denature with SDS and then coat them so all proteins have (-) charge 

  • Eliminates the effect of shape/charge density 

• Load onto polyacrylamide gel

• Apply current through get (all (-) proteins move towards the positive end based on their molecular weight

  • Small proteins move quick, large move slow 

SDS-PAGE electrophoresis: Western Blot

• Comding SDS-PAGE electrophoresis with protein-specific antibodies in western blot results in directly detecting the target proteins 

• Proteins are seperates from polyacrylaminde gel then transferred to membrane 

• The membrane is incubated with the antibody sollution for target protein so it can be detected 

  • Even if similar structures, it will only identify the target protein