lecture 7 Flashcards
(18 cards)
Protein purification is done by
Tagging the Protein: The gene of interest is expressed with an affinity tag (e.g., His-tag or maltose binding protein, MBP).
Affinity Purification:
- The lysate is passed over a resin with affinity for the tag.
- Non-target proteins are washed away, leaving the tagged protein bound to the resin.
- The target protein is eluted, yielding a purified sample.
Steps for Recombinant Protein Expression
Single Colony Pick: Selecting a single bacterial colony.
Cultivation: Growing the bacteria under controlled conditions.
Induction: Activating protein production using inducers like IPTG (Isopropyl β-D-1-thiogalactopyranoside).
Expression: Optimal conditions for expression vary by protein:
- Incubate at 37°C until OD600 reaches 0.4–0.8.
- Induce with 40-400 µM IPTG.
- Expression times:
3 hours at 37°C
5 hours at 30°C
Overnight at 16-23°C
The Bradford assay
Common method for protein quantification.
Bradford Assay:
Principle: This colorimetric assay is based on the absorbance shift of the dye Coomassie Brilliant Blue G-250 when it binds to proteins.
Measurement: The absorbance change is used to determine the protein concentration in the sample.
Microsome
These are small vesicles formed from fragments of the endoplasmic reticulum during cell homogenization.
High in cytochrome P450 enzymes, important for drug metabolism.
Differential Centrifugation:
Different membrane fractions (e.g., microsomes) can be isolated based on their size and density.
Preparative purification
Preparative purifications aim to produce a relatively large quantity of purified proteins for subsequent use.
Examples include the preparation of commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin).
Analytical purification
Analytical purification produces a relatively small amount of a protein for a variety of research or analytical purposes.
Example include identification, quantification, and studies of the protein’s structure, post-translational modifications and function.
Pepsin and urease were the first proteins purified to the point that they could be crystallized.
SEC
Size Exclusion Chromatography (SEC)
Principle: Separation based on molecular size. Large molecules elute first because they cannot enter the pores of the stationary phase, while smaller molecules take longer to elute.
Ion Exchange Chromatography
Principle: Separates proteins based on charge using a charged resin (cation or anion exchange).
Cation Exchange: Binds positively charged proteins.
Anion Exchange: Binds negatively charged proteins.
Procedure:
- Equilibration with counter ions.
- Sample application and washing.
- Elution with a salt gradient (increasing ionic strength).
- Regeneration of the resin.
Affinity Chromatography
Affinity Chromatography
Principle: Utilizes specific interactions between a protein and its binding partner (e.g., His-tag and metal ion).
The target protein is expressed with an affinity tag (e.g., His-tag, GST-tag).
The sample is passed through a column with immobilized ligands specific to the tag.
Elution is achieved by changing buffer conditions (e.g., pH shift, salt gradient, or competition with free ligands).
blotting
transfer of macromolecules, such as nucleic acids and proteins, to solid-phase membranous support
Q: What is the purpose of Polyacrylamide Gel Electrophoresis (PAGE)?
A: PAGE separates proteins based on size using a gel matrix and an electric field, where smaller proteins move faster through the gel.
Q: What is SDS-PAGE and why is SDS used?
A: SDS-PAGE is a type of PAGE that uses SDS (sodium dodecyl sulfate) to denature proteins, giving them a uniform negative charge and allowing separation solely by size.
What role does the stacking gel play in SDS-PAGE?
A: The stacking gel has a lower pH and larger pores, aligning proteins into a tight band before entering the separating gel for size-based separation.
What is Western blotting used for?
A: Western blotting is used to transfer and detect specific proteins separated by SDS-PAGE onto a membrane (PVDF or nitrocellulose).
What are the main steps of a Western blot procedure?
A: 1. Protein separation (SDS-PAGE) 2. Transfer to membrane 3. Blocking non-specific sites 4. Antibody probing (primary and secondary antibodies) 5. Detection and visualization
What is the purpose of the blocking step in Western blotting?
A: Blocking prevents non-specific antibody binding by covering the membrane with proteins like BSA or non-fat milk.
What is the role of the primary and secondary antibodies in Western blotting?
A: The primary antibody binds to the target protein, while the secondary antibody, conjugated to a detection enzyme, binds to the primary antibody for visualization.
