Integrated Solutions for Genomics

1
Overview of 2DE
Complex mixture of proteins
Denature and solubilize in solution (Sample prep)
Separate proteins by charge in first dimension
(IEF)
Separate proteins by size in second dimension
(SDS-PAGE)
Individual proteins isolated as distinct protein
features within a gel matrix
2
Sample Preparation
GSI products
Procedure
Disrupt tissues and cell membrane Mechanical
force and detergents
Sample Buffer II RNAse and DNAse
Disrupt complexes and linearize
proteins chaotropic agents (Urea) reducing agents
(DTT) detergents
Rehydration buffer IPG loading buffer Sample
buffer III
Eliminate highly abundant proteins
Cibachron dye mini-columns
Remove salt from fractionated or affinity
purified samples
Dialysis kits and buffers
3
Isoelectric Focusing- Separation by Charge
Cathode
Anode
-

pH
3 4 5 6 7 8 9 10
Acidic
Basic
Proteins are amphoteric (contain acidic and basic
residues)
For every protein there is a pH at which its net
charge is 0. This is its isoelectric point (pI)
Above its pI, a protein has an overall negative
charge and will migrate toward the positively
charged anode
Below its pI, a protein has an overall positive
charge and will migrate toward the negatively
charged cathode
At its pI, a protein does not move (focuses into
a single band)
4
Final Result of IEF
BPB
Acidic
Basic
Proteins focused into distinct bands
5
Methods for isoelectric focusing
Carrier-ampholyte tube gels pH gradient created
by discontinuous buffering system and carrier
ampholytes Immobilized pH Gradient(IPG)
strips pH gradient fixed in gel by covalently
linking amphoytes to acrylamide when gel gradient
is poured.
6
Investigator Tube Gel Apparatus
Capacity 15 analytical or 8 preparative tubes
7
Investigator IPG pHaser
Runs up to 10 IPG strips
Compatible with all brands of IPG strips
8
Tube gels vs IPG strips
Benefits of tube gels No rehydration step- saves
one day. May be better for some
proteins-membrane, hydrophobic.
Benefits of IPG strips Immobilized pH gradient
eliminates cathodic and anodic drift. Higher
volume of sample can be loaded. Less likely to
become damaged in 2DE procedure. Less labor
involved.
9
Second dimension SDS Page- Separation of proteins
by mass
Coat focused proteins with SDS
GSI products Equilibration buffers I and II
Place strip/tube directly onto 2nd D gel
GSI products 2-D Running System-tank, power
supply, chiller, precast slab gels, gel casting
reagents, premixed buffers
Negatively charged proteins migrate toward anode
Anode
10
Typical results following 2DE
High MW
Low MW
Acidic Proteins
Basic Proteins
11
Investigator 2DE Electrophoresis System
Peltier chiller Programmable Power
Suppy Capacity 5 single or 10 double gels
Single power supply runs tube gels, IPG strips
and slab gels
12
Detection of protein features
Detection limits
Staining method
300ng 30ng 5ng 5ng 1ng
Standard Coomassie Colloidal Coomassie Fluoresce
nt Dyes Non-destructive Silver Destructive
Silver
13
Standard Coomassie Staining
General method Add stain with fixative and
incubate 4hrs to overnight. Destain in 40
Methanol, 10 acetic acid. Benefits Easy and
consistent Mass-spec friendly Disadvantages Least
sensitive stain Requires long incubation times
and destaining
14
Colloidal Coomassie Staining
General Method Incubate gel in colloidal solution
for minutes or hours Destain with water (if
required). Benefits Mass spec friendly Very
fast Environmentally friendly/ less
hazardous More sensitive than standard
coomassie No special visualization
requirements Disadvantages Detection limit 10-50
times lower than fluorescent dyes or silver.
15
Staining using Fluorescent dyes
General method Incubate in dye for 1 hour to
overnight Destain (if desired) Benefits Fast and
easy to use Non-toxic Very sensitive Linear over
a broad range (ng to mg) Mass spec
friendly Disadvantages Requires UV source for
visualization
16
Pros and Cons of Silver Staining
Benefits Most sensitive stain (when
gluteraldehyde is used) Disadvantages Long and
tedious procedure Labor intensive Hazardous Very
sensitive to wash and development times Linear
over a very narrow range Protein specific
staining (some do not stain or stain
negatively) Most sensitive method (destructive)
not compatible with mass spec
17
How much sample should I load?
The amount of sample required depends on
both the staining method, AND the complexity of
the sample
Examples If the sensitivity for Coomassie
equals 40-50ng per feature, then 1,000 features
could be detected starting with 50-100ugs
sample. To visualize all the features in a sample
containing 10,000 features you need to start
with 500ug-1mg total protein. If the
sensitivity for silver equals 1-5ng per feature,
then 1000 features could be detected starting
with 1-5ug sample. To visualize all the features
in a sample containing 10000 features you need
to start with 10-50ug total protein. Note
These examples assume that all proteins in the
sample are present in equal amounts, not the case
in real life.
18
Finding what youre looking for
The simple eukaryote, yeast, has approximately
6000 genes but can produce over 12,000 protein
features due to post-transcriptional and post
translational modifications. To see a rare
protein within a crude extract, 20-2000mgs of
total protein would need to be loaded onto the
1st dimension gel.
Therefore
2DE of crude fractions using broad range IEF gels
can only provide information on relatively
abundant proteins (high copy number). If you want
to detect moderate to rare proteins you must
reduce the complexity of the sample or limit the
scope of the search.
19
Methods for finding the interesting proteins
Prefractionate- Reduce the complexity of each
sample by fractionating into nuclear,
cytoplasmic, mitochondial, microsomal or other
distinct compartments. Remove highly abundant
proteins Purify complexes- Enrich for specific
activities, or complex components. Use narrow
range IEF gels- Increase the amount of sample you
can load on a gel while increasing resolution
within narrow PI ranges (3-6, 5-7,6-8,7-9). The
use of zoom gels (one point pH spread) allows
loading of up to 40mgs starting sample.