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. Disrupt tissues and cell membrane Mechanical force and detergents
Sample Preparation
Procedure
GSI products
Disrupt tissues and cell membrane
Mechanical force and detergents
Non-ionic and zwitterionic detergents
Sample Buffer I
Remove non-protein components
Centrifugation and degrading enzymes
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 + -
Anode
Cathode + - pH
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. Proteins focused into distinct bands
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 Compatible with all brands of IPG strips
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
Standard Coomassie
Colloidal Coomassie
Fluorescent Dyes
Non-destructive Silver
Destructive Silver
300ng
30ng
5ng
1ng

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 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 ugs 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 features you
need to start with ug 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 you’re 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, mgs 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.
2-D gel of sample before (left) and after (right) treatment with albumin depletion kit.