Protein Denaturation

denature: loss of structure due to

protein unfolding
unfolding leads to loss of function

Folded

Unfolded

Sodium Dodecyl Sulfate (SDS)

strongly denaturing detergent
disrupts 2o, 3o, and 4o structures
binds and confers negative charge to protein
charge is proportional to mass

Polyacrylamide gel electrophoresis can

occur under both denaturing and reducing
conditions

Polyacrylamide Gel Electrophoresis (PAGE)

Molecules are separated by size in an
electric field
Smaller molecules migrate more rapidly
through porous gel matrix

Acrylamide Polymerization

Polyacrylamide is a
useful polymer for gel
electrophoresis because
both the concentration
and cross-linking of the
gel can be controlled

Proteins are detected in PAGE gels by

staining or autoradiography

Isoelectric Focusing relies on the migration

of charged proteins in an electric field

Immunoblotting

aka Western Blotting

use Ab to detect protein after
electrophoresis

1. Protein electrophoresis
2. Transfer proteins to membrane
- electrophoretically
- MeOH to reduce SDS
3. Incubate membrane with antibody
4. Extensive washing
5. Detect bound antibody
- radiolabeled 2nd Ab or protein A
- ELISA

Western blotting with antibodies to detect

specific proteins

Antibodies
Analytical Techniques
Utilizing Antibodies:
flow cytometry
gel electrophoresis
immunoprecipitation (IP)
immunoblotting
microscopy
immunofluorescence (IFA)
electron microscopy
ELISA
antibodies bind proteins with
high specificity and affinity
affinity chromatography
analytical techniques

CNBr

Antibodies can be generated against

protein epitopes

Antibodies can bind proteins with very

high affinities

Polyclonal and monoclonal antibodies

The Enzyme-Lined Immunosorbent Assay

(ELISA)

Conventional ELISA
bind protein to membrane or
96-well microplate
neg. (and pos.) controls
purity?
incubate with 1o and 2o
antibodies
use soluble chromogenic
substrates in 96-well plates
quantify Ag or Ab

 measure absorbance with

ELISA microplate reader

Screening Expression Libraries

prepare library in expression vector
cDNA better than gDNA
6X possible reading frames
screen with protein based probe
antibody/western blot
ligand binding or other activity

gt11

Indirect Immunocytochemistry

Stain for DNA.

What is fluorescence?
Energy of light is inversely proportional
to the wavelength. For example, violet
light has higher energy than red light.
Fluorescent molecules are special
molecules that absorb incident light of
a particular wavelength then emit light
of a longer wavelength. The absorption
of light causes electrons in the
molecule to become highly energetic.
When the highly energetic electrons
return to their original state, they
emit light. Wave length of incident
light must be shorter than the
wavelength of emitted light since some
energy is unavoidably lost as heat.

Fluorescence Microscope

Fluorescence image of a cell in mitosis labeled w 3 tags:

Green: fluorescein-labeled anti-tubulin (MTS)
Red: anticentromere
protein
Blue: DAPI
(DNA)

Images of Conventional (A) vs Confocal (B) Fluorescence

(Drosophila embryo stained w fluorescent probe against
actin)

no FBS

control

MBF1
DAPI
Actin

Doxorubicin

Actinomycin D

Protein

DNA + Protein

DNA

DAPI

MBF1 in Drosophila larval tissues

Testis

Imaginal discs

CNS, L2
Salivary glands

Gut

Specific markers
for specific cells