1. 2.1. Two-Dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties, usually isoelectric points and molecular weight, in two dimensions on 2-D gels.
Isoelectric point, abbreviated as pI, is the pH at which a molecule containing two or more ionizable groups is electrically neutral.
Carrier ampholytes (CA) are small, soluble molecules with both positive and negative charge groups. They are used as a carrier for the protein separation in 2-DE.

2. Immobilized pH gradient (IPG) is a pH gradient generated by a limited number (6-8) of well-defined chemicals which are co-polymerized with the acrylamide matrix.
Codon bias index (CBI) is a measure of codon bias, it measures the extent to which a gene uses a subset of optimal codons. In a gene with extreme codon bias, CBI will equal 1.0, in a gene with random codon usage CBI will equal 0.0.

3. Key principles of 2-DE:
Proteins differ from each other in terms of their mass and charge.
Both these properties can be used to separate proteins by gel electrophoresis.
The successive application of both techniques in perpendicular directions (two dimensions) provides maximum separation and allows thousands of proteins to be resolved.
Staining the gel reveals the positions of individual proteins as spots or smudges. These can be picked and analysed by mass spectrometry.
There are tens of thousands of proteins in a cell, differing in abundance over six orders of magnitude.
2-DE is not sensitive enough to detect the rare proteins and many proteins will not be resolved. Splitting the sample into different fractions is often necessary to reduce the complexity of protein mixtures prior to 2-DE.

4. Figure 2.1. The two-dimensional electrophoresis.

5. Figure 2.2. A 2D-PAGE map of Escherichia coli DH10B with glucose.

6. 银 染 色
考马斯亮兰染色
蛋白质丰度
(拷贝数/细胞)
蛋白质量(mg)
细胞数 10
20.073
1.2 x 109
2007.3
1.2 x 1011
100
2.007
1.2 x 108
200.7
1.2 x 1010
1000
0.201
1.2 x 107
20.1
10000
0.020
1.2 x 106
2.0
100000
0.002
1.2 x 105
0.2
There are about 50 to 75 trillion cells in the human body, although these estimates vary depending on the source. Some estimates even go as far as there being 100 trillion cells in the human body.
The minimum amount of proteins is 1ng for detection by the silver staining and 100ng by the Coomassie Brilliant Blue staining.

7. A problem for analyzing low-abundant proteins: They are shielded by high-abundant proteins
The resolution ---- Sequential extraction of proteins:
According to proteins’ solubility;
According to proteins’ location.
Figure 2-2 (page 29)

8. Improvements of IPG:
pH is more precise;
pH is much wider.

9. Limitations of 2-DE:
Membrane proteins
Extremely acidic / basic proteins
Rare proteins
Reproducibility
Large-scale
Automation
(page 31-32)

10. D Chromatography
In 2-D chromatography, different chromatographic techniques are essentially combined either offline or online to achieve a higher degree of separation.
Affinity chromatography - Reverse phase chromatography
Size exclusion chromatography - Reverse phase chromatography
Size exclusion chromatography - Ion exchange chromatography
Ion exchange chromatography - Reverse phase chromatography

12. 2.3 Preparation Of Protein Samples For 2-D Electrophoresis
The General Considerations of the Sample Preparation
The General Methods to Prepare the Samples
Fractional Extraction of Proteins
Protein Extraction from Subcellular Components

13. 2.3.1 The General Considerations of the Sample Preparation
How many and what proteins are interested.
Do you need the separated proteins to keep their natural structures?
Do you have a simple, but effective method to disrupt sample cells?
Are the necessary components included in your lysis buffer?

14. 2.4. The General Methods to Prepare the Samples
The mild methods to disrupt cells:
Use a low osmotic solution.
By freeze-thaw cycle.
Use detergents.
Use lytic enzymes.
The stronger methods to disrupt cells:
Use supersonic.
Use a French pressure cell press (French press).
By grinding.
By a blender.
Use glass beads

15. To protect the sample from protease:
Low temperature
Denaturants, such as urea, TCA, and SDS et al.
Higher pH (>9.0)
Protease inhibitors
Make sure that the subsequent proteolytic reaction is not affected by the employed protease inhibitors.

16. Inhibitors
Protease Class
Stock Conc.
Working Conc.
Attention
PMSF
Serine proteases (Irreversible)
100 mM in methanol
1 mM
PMSF has a half-life in water of minutes. It should be added just before the cell lysis. PMSF is not very soluble in water and should be kept at -20°C in dry methanol/propanol.
Phenylmethanesulfonyl fluoride
AEBSF
100 mM in H2O
Water-soluble, non-toxic alternative to PMSF.
4-(2-Aminoethyl)benzenesulfonylfluoride
EDTA, EGTA
Metalloproteases
500 mM in H2O
Benzamidine
Serine proteases
TLCK, TPCK
Serine & Cysteine proteases
100 mM in DMSO/Ethanol
0.1~0.15mM
Tosyl-L-lysine chloromethyl ketone (TLCK)
Tosyl-L-phenylalanine chloromethyl ketone (TPCK)
Aprotinin
1 mg/ml in H2O
0.6~2.0 mg/ml
Bestatin
Aminopeptidase
1~10 μM
Pepstatin A
Acid proteases,
(Competitive and reversible)
1 µg/ml
Leupeptin
(Reversible)

17. To precipitate proteins:
Ammonium sulphate
Trichloroacetic acid (TCA)
Acetone
Acetone and TCA together (10% of acetone in TCA)
Protein precipitation from phenol phase with Ammonium acetate

18. To clean the sample for 2-DE:
Desalt
Endogenous molecules, such as nucleotides, phospholipids et al.
Detergents
Nucleic acids
Polysaccharides
Lipids
Phenolic constituentes

19. The components of the extraction buffer:
Detergents
Urea
DTT, β-Mercaptol Ethanol (β-ME), tributyl phosphine (TBP)
Carrier ampholytes and IPG buffer

20. Table 3.1. Properties of Common Detergents
Description
Aggregation Number
Micelle MW MW
CMC (mM)
Dialyzable
Triton X-100
Nonionic
140
90,000
647
0.24 No
Triton X-114 —
537
0.21
NP-40
149
617
0.29
Brij-35 40
49,000
1225
0.09
Brij-58 70
82,000
1120
0.077
Tween-20
1228
0.06
Tween-80 60
76,000
1310
0.012
Octyl Glucoside 27
8,000
292
23-25
Yes
Octylthio Glucoside
308 9
SDS
Anionic 62
18,000
288
6-8
CHAPS
Zwitterionic 10
6,149
615
8-10
CHAPSO 11
6,940
631

21. A commercial extraction buffer from MBI:
50mM TRIS-HCl, pH 7.5
105mM NaCl
1% NP-40
1% sodium deoxycholate
0.1% SDS
2mM EDTA

22. Total Protein Extraction from Animal Tissues
Prepare 10mg of tissue per 1ml the extraction buffer and put the tissue in the pre-chilled buffer.
Homogenize using glass homogenizer or tissue tearer on ice.
Put the homogenate on ice for 10min.
Centrifuge at 12000rpm, 4℃ for 20min and transfer the supernatant into new tube.
Store at –20℃.

23. 2.5. Fractional Extraction of Proteins
The combined three-step extractions:
Use a hydrophilic buffer
Use a hydrophobic buffer
Combinatorial detergents
Figure 3-2 (page 58)

24. Figure 3.1. Proteins were extracted according to the phenol extraction method.

25. Figure 3.2. Proteins were extracted according to the phenol extraction method. (a) Clean protein sample. (b) Contaminated protein sample with traces of extraction buffer in the collected phenol phase; contaminants lead to horizontal streaking (incomplete isoelectric focusing), vertical streaking and the disappearance of distinct spots.

26. 2.6. Protein Extraction from Subcellular Components
The advantages:
Localization
Reduced protein numbers both in type and amount
The lysis buffer:
Tris-HCl 10mM pH 7.4, NaCl 50mM, EDTA 5mM
Triton X-100 1%, SDS 0.05%
NaF 50mM, Na3VO4 100μM
beta-glycerophosphate 10mM, sodium pyrophosphate 10mM, phospho-serine 1mM, phospho-threonin 1mM, phospho-tyrosine 1mM
leupeptin 20μg/ml, bacitracine 500μg/ml, trypsin soybean inhibitor 50μg/ml and PMFS 100μg/ml

27. A procedure of preparing a sample for 2-DE:
Start with 1g of tissue washed in cooled PBS
Use liquid nitrogen to fast freeze the sample
Grind the tissue with a mortar and pestle
Place the now powdered tissue into 10 ml lysis buffer
Using a polytron, homogenize the ground tissue and lysis buffer (one burst for20 seconds)
Centrifuge for 15 minutes at 500g at 4 ℃
Remove and keep the supernatant
Homogenize the pellet again with additional 5ml lysis buffer (20 seconds)
Pool both supernatants
Centrifuge the supernatants at 45,000g for 15 minutes at 4 ℃
Wash the pellet twice in lysis buffer
Resuspend the pellet in resuspension buffer
Store at -80℃

28. 2.7 Separation Of Cellular Proteins By 2-D Electrophoresis
Overview
Isoelectric Focusing
SDS-PAGE
Detecting Proteins in Gel
Improvement to Be Made

29. 2.8. Overview The chemicals used in PAGE: Two references:
Acrylamide (Table 4-1, page 69)
N,N’-methylene bisacrylamide (Table 4-1, page 69)
Ammonium persulfate
Tetramethyl ethylenediamine (TEMED)
Two references:
Total solids content (T) T = (a + b) / m
Ratio of cross-linker (C) C = b / (a + b)

30. Figure 4.1. The monomers and their polymerization

31. 2.9. Isoelectric Focusing Synthetic carrier ampholytes:
Poor reproducibility
Electroendosmosis is an osmosis under the influence of an electronic field as in electrophoresis.
Focusing problem for basic proteins
Low mechanical stability
Immobilized pH gradient:
Table 4-2 (page 74)

32. Figure 4.2. APGphor from APB

33. Sample application method:
Sample can be applied either by including it in the rehydration solution (rehydration loading) or by applying it directly to the rehydrated IPG strip via sample cups, sample wells, or paper bridge.
Usually rehydration loading is preferable.
Advantages of rehydration loading:
Rehydration loading allows larger quantities of protein to be loaded and separated.
Rehydration loading allows more dilute samples to be loaded.
Because there is no discrete application point, this method eliminates the formation of precipitates at the application point that often occur when loading with sample cups.
The rehydration loading method is technically simpler, avoiding problems of leakage that can occur when using sample cups.

34. Components of the rehydration solution:
A typical solution generally contains urea, non-ionic or zwitterionic detergent, dithiothreitol (DTT), CA, or IPG Buffer appropriate to the pH range of the IPG strip and dye.
Urea solubilizes and denatures proteins, unfolding them to expose internal ionizable amino acids. Commonly 8 M urea is used, but the concentration can be increased to 9 or 9.8 M if necessary for complete sample solubilization. Thiourea, in addition to urea, can be used to further improve protein solubilization.
Detergent solubilizes hydrophobic proteins and minimizes protein aggregation. The detergent must have zero net charge—use only nonionic and zwitterionic detergents. CHAPS, Triton X-100, or NP-40 in the range of 0.5 to 4% are most commonly used.
Reductant cleaves disulfide bonds to allow proteins to unfold completely. DTT or DTE (20 to 100 mM) are commonly used. 2-Mercaptoethanol is not recommended, because higher concentrations are required, and impurities may result in artifacts.
IPG Buffer or carrier ampholyte mixtures improve separations, particularly with high sample loads. Carrier ampholyte mixtures enhance protein solubility and produce more uniform conductivity across the pH gradient without disturbing IEF or affecting the shape of the gradient.

35. Table 4.1. Suitable sample loads for silver and Coomassie staining using rehydration loading

37. 2.10. SDS-PAGE Equilibration solution components:
Equilibration introduces reagents essential for the second-dimension separation.
Equilibration buffer (50 mM Tris-HCl, pH 8.8) maintains IPG strip pH in a range appropriate for electrophoresis.
Urea (6 M) together with glycerol (30%) reduces the effects of electroendosmosis by increasing the viscosity of the buffer and improves transfer of protein from the first to the second-dimension.
DTT preserves the fully reduced state of denatured, unalkylated proteins.
SDS denatures proteins and forms negatively charged protein-SDS complexes.
Iodoacetamide alkylates thiol groups on proteins, preventing their reoxidation during electrophoresis. Protein reoxidation during electrophoresis can result in streaking and other artifacts.
Bromophenol blue allows monitoring of electrophoresis.

38. Equilibration steps:
Make the second-dimension vertical gel ready for use prior to IPG strip equilibration.
Prepare equilibration solution: 2% SDS, 50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 0.002% bromophenol blue.
Just prior to use, add 100 mg DTT per 10 ml SDS equilibration buffer.
Place the IPG strips in individual tubes with the support film toward the wall. Add 10 ml of the DTT-containing solution to each tube. Cap the tube, and place it on its side on a rocker. Equilibrate for 15 min.
A second equilibration may be performed with an iodoacetamide solution (without DTT). Prepare a solution of 250 mg iodoacetamide per 10 ml SDS equilibration buffer.
Add 10 ml of solution per tube. Cap the tube, place it on its side on a rocker, and equilibrate for 15 min.

39. Select the gel percentage:
Single percentage gel versus gradient gel.
When a gradient gel is used, the overall separation interval is wider. In addition, sharper bands result because the decreasing pore size functions to minimize diffusion. However, a gradient gel requires more skill to cast.
Single percentage gels offer better resolution for a particular Mr window. A commonly used second-dimension gel for 2-D electrophoresis is a homogeneous gel containing 12.5% total acrylamide.
Stacking gels are not necessary for vertical 2-D gels.
Whether single percentage or gradient, the appropriate percentage gel is selected according to the range of separation desired.

40. 2.11. Detecting Proteins in Gel
The following features are desired:
High sensitivity
Wide linear range for quantification
Compatibility with mass spectrometry
Low toxicity and environmentally safe
Table 4-4 (page 82)

41. The mainly used methods:
Autoradiography and fluorography are the most sensitive detection methods (down to 200 fg protein).
For autoradiographic detection, the gel is simply dried and exposed to X-ray film.
Silver staining is the most sensitive non-radioactive method (below 1 ng). Silver staining is a complex, multi-step process utilizing numerous reagents for which quality is critical.
Coomassie staining, although 50- to 100-fold less sensitive than silver staining, is a relatively simple method and more quantitative than silver staining. Coomassie blue is preferable when relative amounts of protein are to be determined by densitometry.
Negative Zinc-Imidazole staining has a detection limit of approx. 15 ng protein/spot and is well compatible with mass spectrometry, but it is a poor quantification technique.
Fluorescent labelling and fluorescent staining with SYPRO dyes have a sensitivity between Coomassie and modified silver staining. This technique require fluorescence scanners, but they are compatible with mass spectrometry and show a wide dynamic range for quantification.

42. 2.12. Improvement to Be Made There are problems as following:
Detection of low abundant proteins
Separation of extremely acidic/basic proteins
Too large proteins (Mr > 100 kDa)
Membrane proteins
Automation