Schematic representation of a series of2-D gels

showing different stages of a pmteome.

2D electrophoresis

Protein identification by peptide mass fingerprint

Sample preparation
1. Cancel different oxidations steps.
2. Prevent protein aggregates.
3. Deactivate proteases.
4. Convert all proteins into single conformations.
5. Prevent protein modifications.
6. Get hydrophobic proteins into solution and keep them in s
tion.
7. Cleave disulfide and hydrogen bonds; uncoil the polypept
to expose all buffering groups to the medium.

lysis buffer lysis

9 M (mol/L) urea, 4 % CHAPS, 1 % DTT , 0.8 % carrier
ampholytes, 0.002 % bromophenol blue

lysis

1
More vigorous lysis methods

More vigorous lysis methods

Sample preparation
1. Cancel different oxidations steps.
2. Prevent protein aggregates.
3. Deactivate proteases.
4. Convert all proteins into single conformations.
5. Prevent protein modifications.
6. Get hydrophobic proteins into solution and keep them in s
tion.
7. Cleave disulfide and hydrogen bonds; uncoil the polypept
to expose all buffering groups to the medium.

2
Deactivate proteases
Sample preparation
1. Cancel different oxidations steps.
2. Prevent protein aggregates.
3. Deactivate proteases.
4. Convert all proteins into single conformations.
5. Prevent protein modifications.
6. Get hydrophobic proteins into solution and keep them in s
tion.
7. Cleave disulfide and hydrogen bonds; uncoil the polypept
to expose all buffering groups to the medium.
lysis buffer
9 M (mol/L) urea, 4 % CHAPS, 1 % DTT , 0.8 % carrier
ampholytes, 0.002 % bromophenol blue
lysis
Deactivate proteases
Sample preparation
1. Cancel different oxidations steps.
2. Prevent protein aggregates.
3. Deactivate proteases.
4. Convert all proteins into single conformations.
5. Prevent protein modifications.
6. Get hydrophobic proteins into solution and keep them in s
tion.
7. Cleave disulfide and hydrogen bonds; uncoil the polypept
to expose all buffering groups to the medium.
lysis buffer
9 M (mol/L) urea, 4 % CHAPS, 1 % DTT , 0.8 % carrier
ampholytes, 0.002 % bromophenol blue
3
 lysis buffer
Sample preparation
9 M (mol/L) urea, 4 % CHAPS, 1 % DTT , 0.8 % carrier
1. Cancel different oxidations steps.
ampholytes, 0.002 % bromophenol blue
2. Prevent protein aggregates.
3. Deactivate proteases.
4. Convert all proteins into single conformations.
5. Prevent protein modifications.
6. Get hydrophobic proteins into solution and keep them in s
tion.
7. Cleave disulfide and hydrogen bonds; uncoil the polypept
to expose all buffering groups to the medium.

lysis buffer Precipitation procedures

9 M (mol/L) urea, 4 % CHAPS, 1 % DTT , 0.8 % carrier
ampholytes, 0.002 % bromophenol blue
lysis

DTT

Concentration
Desalting

Precipitation procedures Precipitation procedures

4
Precipitation procedures Precipitation procedures

Salting out

Precipitation procedures

Patch Denature

Very hydrophobic proteins:

E. coli : IEF in 7 cm IPG 4-7
Membrane proteins do not easily go into solution.
Optimization work is required for hydrophobic protein samples

A: E. coli extract was precipitated with TCA / 8 M urea 7 M urea, 2M thiourea

acetone and resuspended with lysir buffer. B:
Crude extract.

5
Removal of contaminants that affect 2-D result Removal of contaminants that affect 2-D result

Desalting can be performed by DNA and RNA can be removed by

• dialysis • DNAse
• spin dialysis • RNAse
• gel filtration
• precipitation/resuspension

Endogenous small ionic molecules, (nucleotides,

metabolites, phospholipids, etc) can be removed by

TCA/acetone precipitation

2D electrophoresis (二維電泳)

第一維：Isoelectric focussing electrophoresis

Carrier Ampholytes

Carrier Ampholytes

6
Carrier Ampholytes Batch-to-batch variation Immobiline (immobilized pH gradients)

Immobiline (immobilized pH gradients) Immobiline

Immobiline Immobiline

7
Immobiline

Linear vs. non-linear

8
Strip rehydration Strip rehydration (12 hr)

Strip rehydration cup loading

Strip rehydration + in-gel loading

Strip rehydration cup loading

Strip rehydration + in-gel loading

9
Strip rehydration + in-gel loading

Isoelectric focussing electrophoresis

Isoelectric focussing electrophoresis Isoelectric focussing electrophoresis

Step-and-hold

Isoelectric focussing electrophoresis Isoelectric focussing electrophoresis

Step-and-hold Gradient

10
Isoelectric focussing electrophoresis Isoelectric focussing electrophoresis
Step-and-hold
Gradient

Isoelectric focussing electrophoresis Isoelectric focussing electrophoresis

500 V 500 Vhr

1000 V 1000 Vhr
2000 V 2000 Vhr
4000 V 4000 Vhr
6000 V 6000 Vhr
8000 V => total = 100 kVhr

Isoelectric focussing electrophoresis

Focus and overloading 2nd electrophoresis

Underfocusing
Overfocusing

11
Equilibration DTT

iodoacetamide

Equilibration 1-D SDS PAGE 2-D SDS PAGE

Markers

SDS-PAGE: non-gradient SDS-PAGE: non-gradient

12
SDS-PAGE: gradient SDS-PAGE: gradient

SDS-PAGE SDS-PAGE

Troubleshooting Troubleshooting

13
Troubleshooting Troubleshooting

Troubleshooting Troubleshooting

Troubleshooting Troubleshooting

14
Troubleshooting Troubleshooting

Troubleshooting

15