Exam 4 lecture 8 - Techniques/Test

Question 1

What does FRAP Measure?

Answer 1

Lipid Mobility

Lateral diffusion can be demonstrated using
fluorescence recovery after photobleaching
(FRAP)

( laser beam bleach experiment)

Question 2

Thin-layer chromatography (TLC) is used to

Answer 2

separate different kinds of lipids based on their
relative polarities

Question 3

The transition temperature can be measured by

Answer 3

differential scanning
calorimetry

Question 4

Freeze-Fracture

Answer 4

Analysis of Membranes

When a fracture plane splits a membrane into its two layers,
particles the size and shape of globular proteins can be seen
 The E surface is the exoplasmic face, and the P surface is the
protoplasmic face

Question 5

Isolation of Membrane Proteins

Answer 5

Peripheral membrane proteins are usually easy to
isolate by altering pH or ionic strength
 Chelating (cation-binding) agents are also used to
solubilize peripheral membrane proteins
 Lipid-anchored proteins are isolated by
similar means

Question 6

Electrophoresis is a group of techniques that

Answer 6

use
an electric field to separate charged molecules
 How quickly a molecule moves during
electrophoresis depends on both charge and size

Question 7

Two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-
PAGE)

Answer 7

separates proteins in two dimensions, first by charge and
then by size

Question 8

Western blotting

Answer 8

follows electrophoresis and identifies polypeptides

Question 9

Affinity labeling

Answer 9

utilizes radioactive molecules that
bind to certain proteins based on function

Question 9

membrane reconstitution

Answer 9

proteins are extracted
from membranes and separated individually
 The purified proteins are mixed with phospholipids
to form vesicles call liposomes
 Liposomes can be loaded with particular molecules
and tested for their ability to carry out certain

Question 10

X-ray crystallography can be used to determine

Answer 10

the
structure of proteins that can be isolated in
crystalline form

widely used to determine
the three-dimensional structure of proteins

alternative = hydropathy analysis

Question 11

hydropathy analysis

Answer 11

A hydropathy (or hydrophobicity) plot is used for determining the number and location of transmembrane segments in a membrane protein
( used if the ptoein sequence is known0

Question 12

Site-specific mutagenesis

Answer 12

determination of
how certain amino acids affect protein function

Question 12

Radioactive labeling procedures are used to

Answer 12

distinguish between proteins on
inner and outer surfaces of membrane vesicles

Question 13

cell fusion experiments

Answer 13

provide evidence for mobility of some membrane proteins

performed by David Frye and Michael Edidin

Question 14

The Frye and Edidin Experiments

Answer 14

Frye and Edidin fused human and mouse cells and used two fluorescent
antibodies, each with a differently colored dye linked to it
 The anti-mouse antibodies were linked to fluorescein, a green dye; and the
anti-human antibodies were linked to rhodamine, a red dye
 Within a few minutes of fusion, the red and green region proteins began to
intermix