Gel Electrophoresis in Protein Isolation

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Questions and Answers

Why is it necessary to purify proteins?

To produce antibodies

To study protein regulation and protein-protein interactions

To perform structural analysis by X-ray crystallography

All of the above (correct)

What is the primary purpose of homogenization in protein purification?

To isolate proteins that are components of the membrane or subcellular assembly

To remove cell components denser than the desired organelle

To break down cells and release proteins (correct)

To separate proteins based on their sizes

What is the purpose of centrifugation in protein purification?

To break down cells and release proteins

To remove cell components denser than the desired organelle (correct)

To separate proteins based on their sizes

To isolate proteins that are components of the membrane or subcellular assembly

What is the purpose of stabilization of proteins in protein purification?

To preserve protein structure and function Signup and view all the answers

What is the ideal temperature for protein purification?

Low temperature (~ 0°C) Signup and view all the answers

Why is it essential to inhibit proteases during protein purification?

To prevent protein degradation Signup and view all the answers

What is the purpose of using sodium azide during protein purification?

To retard microbial growth Signup and view all the answers

Where can proteins be obtained from?

From whole organisms, organs or tissues, embryos, tissue culture cells, and proteins from expression systems Signup and view all the answers

What is the principle of ion-exchange chromatography?

Attraction between oppositely charged ion-exchangers and analyte Signup and view all the answers

What is the movement of charged particles through an electrolyte when subjected to an electric field called?

Electrophoresis Signup and view all the answers

What is the purpose of the equilibration step in ion-exchange chromatography?

Stabilization of the ion-exchangers with oppositely charged ions in the buffer Signup and view all the answers

What is the stationary phase in gel-filtration chromatography?

Porous beads Signup and view all the answers

What is the purpose of the tracking dye in gel electrophoresis?

To provide a visible marker for the migration of proteins Signup and view all the answers

Which of the following is an example of an anion-exchanger?

Diethylaminoethyl (DEAE) group Signup and view all the answers

Which type of gel electrophoresis is classified based on the gel casting technique?

Vertical Signup and view all the answers

What is the purpose of dialysis in protein isolation?

To separate proteins from small molecules such as salts Signup and view all the answers

What is the purpose of the regeneration step in ion-exchange chromatography?

Preparation of the ion-exchangers for the next round of protein purification Signup and view all the answers

Which of the following chromatography techniques is based on the molecular size of proteins?

Gel-filtration chromatography Signup and view all the answers

Which of the following methods is used to separate proteins based on their ionic charge?

Ion-exchange chromatography Signup and view all the answers

Which reagent is commonly used for salting-out?

Ammonium sulfate Signup and view all the answers

What is the purpose of the elution step in ion-exchange chromatography?

Removal of bound protein from the ion-exchangers Signup and view all the answers

What is the purpose of gel filtration chromatography?

To separate proteins based on their molecular size Signup and view all the answers

What is the purpose of β-mercaptoethanol in SDS-PAGE?

To reduce disulphide bridges Signup and view all the answers

What is the characteristic of the gel in Discontinuous Gel Electrophoresis?

pH differs from one gel to another Signup and view all the answers

What is the direction of movement of cations during electrophoresis?

Towards the cathode Signup and view all the answers

What is the supporting media used for the separation of proteins in gel electrophoresis?

Polyacrylamide Signup and view all the answers

What is the term used to describe the process of decreasing protein solubility by adding salt?

Salting-out Signup and view all the answers

What is the process of increasing protein solubility by adding salt called?

Salting-in Signup and view all the answers

What is the name of the most widely used method in the analysis of complex mixtures of proteins?

Polyacrylamide gel electrophoresis (PAGE) Signup and view all the answers

Which method is used to separate proteins based on their binding specificity?

Affinity chromatography Signup and view all the answers

Which of the following is an example of a cation-exchanger?

Carboxymethyl (CM) group Signup and view all the answers

Which method is used to separate proteins based on their molecular size?

Gel filtration chromatography Signup and view all the answers

What is the basis of protein separation in Isoelectric Focusing?

Isoelectric point Signup and view all the answers

What is the effect of a protein's isoelectric point on its mobility in an electric field?

The protein's mobility becomes zero Signup and view all the answers

What is the purpose of urea in Isoelectric Focusing?

To denature the protein Signup and view all the answers

What is the function of ampholytes in Isoelectric Focusing?

To create a pH gradient Signup and view all the answers

Which type of PAGE is used to separate proteins based on their molecular weight?

SDS-PAGE Signup and view all the answers

What is the difference between Native PAGE and SDS-PAGE?

Native PAGE is used to separate proteins in their native state, while SDS-PAGE is used to separate proteins in their denatured state Signup and view all the answers

Study Notes

Protein Purification

Proteins are isolated from various sources, including whole organisms, organs or tissues, embryos, tissue culture cells, and proteins from expression systems.
Protein purification is necessary to identify the structure and function of the protein of interest, study protein regulation and protein-protein interactions, produce antibodies, and perform structural analysis by X-ray crystallography.

Steps of Protein Purification

Step 1: Solubilization of protein
- A. Homogenization:
  - On a laboratory scale: sonication, French Press, enzymatic or chemical cleavage of cells, and freeze-thawing
  - For larger scale: high-pressure homogenization and bead milling
- B. Centrifugation:
  - Isolation of proteins that are components of the membrane or subcellular assembly
  - Centrifugation speed is adjusted to remove only the cell components denser than the desired organelle
- C. Filtration
Step 2: Stabilization of proteins
- pH: Proteins are placed in buffered solutions at or near their native pH to prevent denaturation or loss of function
- Temperature: Protein purification is normally carried out at low temperature (~ 0°C), while some proteins are thermally stable at high temperatures
- Inhibition of proteases
- Retardation of microbes that can destroy proteins (e.g., using sodium azide)
Step 3: Isolation of proteins
- Characteristic-based isolation methods:
  - Solubility: salting out, salting in
  - Molecular size: dialysis, gel filtration chromatography
  - Ionic charge: ion-exchange chromatography, gel electrophoresis, isoelectric focusing
  - Binding specificity: affinity chromatography

Ion-Exchange Chromatography

Stationary phase: ion-exchangers (e.g., carboxymethyl (CM) group, diethylaminoethyl (DEAE) group)
Mobile phase: buffers of variable concentrations
Principle: Attraction between oppositely charged ion-exchangers and analyte (protein sample)
Steps:
1. Equilibration: Stabilization of the ion-exchangers with oppositely charged ions in the buffer
2. Sample application and wash: Protein bound to the ion-exchangers remains attached while other proteins are removed during wash
3. Elution: Removal of bound protein from the ion exchangers with the help of increased concentration of elution buffer
4. Regeneration: Preparing the ion exchangers for the next round of protein purification

Gel Filtration Chromatography

Also known as molecular exclusion chromatography
Stationary phase: Porous beads (e.g., Sephadex, Sepharose)
Mobile phase: Buffer (e.g., tetrahydrofuran, chloroform, dimethylformamide)