Lecture 3

Question 1

How does fluorescence microscopy work?

Answer 1

1. First barrier filter only lets light of a certain wavelength range through
2. Beam-splitting mirror reflects light below a certain wavelenth towards object, but transmits light from object above that wavelength
3. Second barrier filter cuts out unwanted fluorescent signals –> emits light @ higher wavelength

**light emitted from second filter is at a diff wavelength then when it was transmitted through first filter

Question 2

How does confocal laser scanning microscopy work?

Answer 2

1. Laser shined through dichroic mirror
2. Light reflects off 2nd dichroic mirror onto specimen stained w/ fluorescent dye
3. Light reflects off specimen back through 2nd dichroic mirror
4. Pinhole eliminates all the out-focused light and only allows some light through

Question 3

What organism is green fluorescent protein isolated from?

Answer 3

Jellyfish

Question 4

What is GFP used for?

Answer 4

GFP marker: can fuse DNA encoding GFP onto a gene encoding a protein of interest

Question 5

The gene Src normally is activated by extracellular signals called growth factors. When the growth factors are present, Src instructs the cell to divide. A mutant form of Src is found in many cancer tumors and is active in the absence of growth factors. The normal Src is a:

Answer 5

Proto-oncogene

Question 6

Describe the structure of an IgG antibody.

Answer 6

• 2 heavy chains (Y formation) connected to light chains (1 on either branch of the”Y”) via disulfide bond (3)
• 2 antigen binding sits on ends of light chian

Question 7

What is the F(ab) fragment of an IgG antibody?

Answer 7

Variable region aka the binding site

Question 8

What is the F(c) fragment of an IgG antibody?

Answer 8

Constant region that has exactly the same AA sequence for all the IgGs from that animal

Question 9

How many F(ab) regions does an IgG antibody have?

Answer 9

2 b/c bivalent

Question 10

How many F(c) regions does an IgG antibody have?

Answer 10

1

Question 11

What does the F(ab) fragment bind to?

Answer 11

Small epitope (piece of an antigen) on the protein (antigen) of interest

Question 12

Polyclonal vs. monoclonal antibodies?

Answer 12

• Polyclonal = multiple epitopes

- Monoclonal = highly specific, limited

Question 13

What is an epitope?

Answer 13

Small piece (~6 AAs) on the surface of an antigen (whole protein) that is recognizable by an IgG

Question 14

What is IgG?

Answer 14

Class of antibodies that is in its free form in the blood

Question 15

What does each IgG consist of?

Answer 15

• 2 identical light chains

- 2 identical heavy chains

Question 16

What is a western blot analysis used for?

Answer 16

To see if the protein of interest is present in your sample, how much of it is there, and what the molecular weight of the protein is

Question 17

How does a western blot analysis work?

Answer 17

1. Cells are lysed w/ SDS detergent
2. Lysates subject to SDS-PAGE (gel electrophoresis) to separate all the proteins based on size (small = fast)
3. Proteins are transferred along an electrical current from gel to a membrane
4. Membrane is probed w/ an antibody specific to the antigen of interest

Question 18

What is SDS? Function?

Answer 18

Strong ionic detergent used to solubilize membranes, denature proteins, and provide a uniform (-) charge

Question 19

What kinds of proteins move fastest on the SDS PAGE?

Answer 19

Smaller proteins move faster and farther in the gel

Question 20

Primary vs. Secondary antibody? Function of each?

Answer 20

• Primary: first antibody that recognizes your protein

- Secondary: recognizes Fc region of primary antibody –> amplifies signal

Question 21

What property would you take advantage of to separate the ER from Golgi?

Answer 21

Density of the organelle

Question 22

Antibodies (IgG) are powerful reagents because they bind to a specific antigen via the ___ and to protein A from bacteria via the ___.

Answer 22

• Fab region

- Fc region