Ch. 5

Question 1

What are the steps of protein purification starting with tissues or cells?

Answer 1

1. Cell lysis
2. Protein binding to matrix
3. Washing
4. Elution

Question 2

How should total protein and specific activity change as you purify?

Answer 2

Total protein should decrease as activity increases

Question 3

What are the different column chromatography techniques?

Answer 3

• Size exclusion/gel filtration
• Ion exchange
• Affinity

Question 4

Size exclusion/gel filtration chromatography

Answer 4

Separates proteins based on size

Question 5

Ion exchange chromatography

Answer 5

Separates proteins based on net charge
- Uses 2 matrices: DEAE and CMC
- DEAE: positively charged, anion-exchange matrix
- CMC: negatively charged, cation-exchange matrix

Question 6

Affinity chromatography

Answer 6

Separates proteins based on binding affinity to ligands

Question 7

SDS PAGE

Answer 7

Way to separate denatured proteins by charge and size (final step to determine purity)

Question 8

Western blot (immunoblot)

Answer 8

Used to detect proteins separated by gel electrophoresis
- Uses primary (protein-specific) and secondary (detection) antibodies

Question 9

What do SDS and beta mercaptoethanol do in SDS PAGE?

Answer 9

• SDS PAGE adds a negative charge to all the proteins
• Beta mercaptoethanol breaks disulfide bonds

Question 10

What is the role of monoclonal antibodies?

Answer 10

• Homogeneous Ig species that recognize 1 epitope on an antigenic protein

Question 11

What is the role of polyclonal antibodies?

Answer 11

• Heterogeneous mix of Ig proteins that recognize 1 or more epitopes on an antigenic protein

Question 12

What reserach methods use antibodies?

Answer 12

• Western blot (immunoblot)
• Affinity chromatography
• Immunofluorescence
• Immunoprecipitation
• Co-immunoprecipitation
• ELISA

Question 13

ELISA (enzyme-linked immunosorbent assay)

Answer 13

• Identifies low level antigenic proteins
• Direct and indirect ELISA

Question 14

Immunofluorescence

Answer 14

• Used to identify proteins in cells that have been chemically treated in a way that preserves the cell’s architecture
• After washing, cells can be visualized through fluorescence microscopy

Question 15

What are the advantages of NMR vs X-ray crystallography?

Answer 15

NMR advantages
- Good for proteins still in solution
- No need to crystalize
- Good for studying dynamics of proteins during catalysis

X-ray crystallography advantages
- No theoretical size limitation

Question 16

Practice question: What is the correct order to to isolate and organelle from a tissue if the organelle isn’t as dense as the nucleus?

Answer 16

1. Obtain tissue
2. Break open the cells to obtain a homogenate
3. Centrifuge at a low speed
4. Take the supernatant and centrifuge at a great speed for a longer time to obtain a second pellet

Question 17

Practice question: What are the purposes of SDS and beta mercaptoethanol in SDS PAGE electrophoresis?

Answer 17

• SDS gives all the proteins a negative charge
• Beta mercaptoethanol breaks disulfide bonds

Question 18

Practice question: Which technique is the best to access purity of your protein?

Answer 18

SDS PAGE

Question 19

Practice question: DNA was attached to chromatography resin and then the proteins that bind DNA were isolated. This is an example of ___.

Answer 19

Affinity chromatography

Question 20

Practice question: As you purify a protein, you want ___ to increase while ___ decreases.

Answer 20

• Activity
• Total protein