Lecture 15 + 16

Question 1

What are the 3 type of column chromatography and What is column chromatography

Answer 1

• separates proteins based on differential physical or chemical interactions with a solid gel matrix
• ion exchange
• size exclusion (gel)
• affinity

Question 2

Different ways to open up/break cells

Answer 2

Sonification - high sound frequency waves
Shearing - blend up the cells
Mild detergent - breaks the cell membrane

Question 3

What is the process of breaking cells and tissues

Answer 3

• first step in the purification of most proteins is to disrupt tissues and cells in a controlled fashion
• using gentle mechanical procedures, called homogenization, the plasma membranes of cells can be ruptured so that the cell contents are released - 4 commonly used procedures
  - sonification (break cells with high-frequency sound)
  - use a mild detergent to make holes in the plasma membrane
  - cell suspension or tissue (force cells through a small hole using high pressure)
  - shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel
• the resulting thick soup (homogenate) contains large and small molecules from the cytosol, such as enzymes, ribosomes, and metabolites, as well as all the membrane-enclosed organelles

Question 4

Centrifugation

Answer 4

• separates macromolecules on the basis of their density, centrifugal force, and the centrifugation time
• differential centrifugation is often used as an initial step in protein purification

Question 5

Salting out

Answer 5

• most proteins are less soluble in high salt concentrations and will precipitate out of solution
• proteins differ in their solubility, fibrinogen will precipitate our of 0.8M ammonium sulfate whereas serum albumin precipitates out at 2.4M
• salt, ammonium sulfate, is added at a concentration (55%) at which our protein of interest is insoluble
• all other proteins insoluble in 55% (NH4)2SO4 are precipitated out by centrifugation
• the pellet is re-suspended in a physiological buffer

Question 6

Size exclusion (gel)

Answer 6

• separates proteins based on size
• protein mixture is added to column containing cross-linked polymer
• large proteins cannot enter the pores of the gel matrix beads and elute from the column first
• the smaller proteins elute later because they are slowed down by entering the beads

Question 7

Ion exchange

Answer 7

• separates proteins based on charge
• depending on the type of resin (marix) used in the column, charged proteins are differentially retained or flow through the column
• protein mixture is added to column containing cation exchangers
• proteins move through the column at rates determined by their net charge at the pH being used
• with cation exchangers, proteins with a more negative net charge move faster and elute earlier

Question 8

Affinity

Answer 8

• separates proteins based on specific ligand interactions
• the target protein specifically interacts with the ligand that is covalently bound to resin, whereas nonspecific proteins flow through the column without interacting

Question 9

Explain the principles of SDS-PAGE

Answer 9

• technique used to separate proteins based on their molecular weight
• SDS, an anionic detergent denatures proteins and imparts a uniform negative charge allowing them to migrate through a gel matrix towards the positive electrode
• smaller proteins move faster and travel further

Question 10

2D PAGE

Answer 10

• used to separate proteins on the basis of both pl and molecular mass
• a protein mixture is separated in the first dimension by isoelectric focusing (based on their overall charge)
• then the proteins are further separated in the second dimension by standard SDS-PAGE
• protein spots on the 2D gel can be excised from the gel and identified by mass spectrometry

Question 11

2D DIGE

Answer 11

• use the fluorescent dyes Cy3 and Cy5 to distinguish two protein samples run on the same 2D PAGE gel
• a protein unique to one sample shows a single color whereas proteins common to both sample appear as shades of yellow that are a mixture of green and red

Question 12

Distinguish between an analytical technique and a preparative one for protein separation

Answer 12

analytical - A280, SDS-PAGE, 2D gel electrophoresis, immune/Western blot, mass spectroscopy
preparative - centrifugation, salting out, column chromatography

Question 13

Proteomics

Answer 13

• large scale study of proteins, particularly their structures and functions
• refers to the study of the entire set of proteins for an organism or the set of proteins present in a particular cell type under a particular set of conditions
• proteome differs from cell to cell and is constantly changing depending on the interactions of the cell with its environment
• tools: isoelectric focusing, protein arrays and chips, (co-IP), (MS)

Question 14

The proteomics approach

Answer 14

• separate the proteins
• cut protein spots out of gel
• digest (cut up) with protease (trypsin)

Question 15

How can we determine protein structure

Answer 15

• NMR spectroscopy
• X Ray crystallography

Question 16

Antibodies

Answer 16

• proteins made by animals in large quantities in response to the presence of a foreign substance called an “antigen”
• made by immune system
• bind to their antigen with high specificity and affinity
• can be used to identify proteins and their locations in the cell to purify proteins, quantify them, and to identify protein binding partners
• antigen binding site in the Fab domain binds to an epitope
• antibodies bind antigens due to stereochemical complementarity between the antigen binding site and the epitome on the antigen

Question 17

ELISA

Answer 17

1. coat surface with sample (antigens)
2. block unoccupied sites with nonspecific protein
3. incubate with primary antibody against specific antigen
4. incubate with secondary antibody - enzyme complex that binds primary antibody
5. add substrate
6. formation of colored product indicates presence of specific antigen

Question 18

Immunoblotting (Western blots)

Answer 18

• transfer proteins from the gel onto filter membrane , wash the membrane with blocking buffer to decrease nonspecific antibody binding
• incubate the membrane with the primary antibody
• wash and incubate the membrane with the enzyme-linked secondary antibody, then add the enzyme substrate
• the enzyme product identifies the location of the target proteins on the membrane

Question 19

Immunofluorescence

Answer 19

• an antibody-based technique used to identify proteins in cells that have been chemically treated in a way that preserves cell architecture