1. 双向电泳的样品制备
有效避免样品损失

2. -大鼠舌组织 (26 µg)
-pH 3-10, 13cm
-无样品处理.
在一向等电聚焦过程中Bromophenol dye没有发生移动.
Contributed by Janice Cheung, GE-Healthcare Malaysia

3. -大鼠舌组织 (26 µg)
-pH 3-10, 13cm
-样品经过 Ettan™ 2DClean Up Kit处理.
-SDS-PAGE胶最后用 Plus One Silver Staining kit染色
Contributed by Janice Cheung, GE-Healthcare Malaysia

4. 样品的准备步骤 Cell disruption Protein precipitation Solubilization
Protection against protease activities
Removal of
nucleic acids
lipids
salts, buffers, ionic small molecules
insoluble material
Cell disruption method is dictated by the cell type and tissue. Cell disruption should be complete, but without protein modification.
Often, the protein in the lysate is precipitated. This concentrates protein and can aid in sample clean up by leaving behind impurities that might otherwise interfere with 2-D separation or visualization.
Proteins in the sample must be soluble for electrophoresis. This is done with urea and detergent. The detergent must be non-ionic or zwitterionic, but SDS can be used if necessary if it is subsequently diluted with non-ionic detergent (final SDS:non-ionic detergent ratio must be 1:8 or less).
Protection from proteases is particularly important in samples that will sit at room temperature overnight for rehydration. 8 M urea will inhibit most proteases.
Nucleic acids and lipids are both negatively charged and can cause problems with separation. They can also cause silver staining problems. DNase and RNase can be used to remove nucleic acids. Organic precipitation or extraction removes lipids.
Other ionic impurities can complicate separation and can be removed by precipitation or dialysis.
Best results are obtained when insoluble material is removed by ultracentrifugation.

5. 在样品制备前需要考虑的问题 Is the sample from cells or solid tissue?
Is pre-fractionation desired?
What kind of interfering substances are present?
Quality of separation vs. total protein representation
Solid tissues require grinding or mechanical disruption. Cell cultures can be lysed with detergent, freeze-thaw or osmotic lysis if no cell wall is present. Microorganisms with cell walls require sonication or enzymatic lysis.
Sometimes just a single subcellular fraction is desired (e.g. plasma membrane, mitochondria, flagella).
Sometimes the sample is separated into solubility fractions for separate analysis (Water soluble, Triton-X-100 soluble, SDS soluble)
Any step taken to remove an interfering substance can potentially remove proteins too. These steps need to be carefully considered if the aim is to analyze as many different proteins as possible.

6. 细胞破碎的方法 Freeze-thaw or osmotic lysis Detergent lysis Sonication
Enzymatic lysis
French pressure cell
Grinding (mortar and pestle)
Mechanical homogenization
Freeze thaw and osmotic lysis are very gentle techniques that are only effective on easily-lysed cells. These techniques may be employed if complete cell disruption is not desired (e.g. when only cytoplasmic proteins, or mitochondrial proteins are being analyzed).
Detergent lysis can be effective on cultures of cells that have no hard cell wall. Detergent/urea mixtures used for IEF can be effective as lysis solutions, resulting in a lysate that can often be subjected to IEF with now further treatment.
Sonication breaks open cells with high frequency sound and is effective on cells with cell walls (e.g. bacteria, yeasts, algae). Sonication can also be useful in later stages of sample prep, such as resuspending and resolubilizing precipitated protein. A sonicator can be a good investment for a lab wanting to get into 2-D.
A so-called French press squeezes a cell suspension through a small orifice at very high pressure. The resulting shear force breaks open the cells.
Solid tissues can be ground with a mortar and pestle. Often the tissue is first frozen solid with liquid nitrogen and then ground to a powder. This avoids proteolysis and other kinds of protein degradation as the tissue disruption is done at very low temperature.
Solid tissues can also be disrupted in a blender or with any number of different mechanical homogenizers that are available.

7. 现在常用在样品预分离的方法 By solubility
sequential extraction with increasingly strong solubilizing agents (???)
By chromatography
e.g. hydrophobic interaction (???)
By centrifugal separation of subcellular components
e.g. mitochondria, nuclei, cytosol
By affinity
Immunoprecipitation of complexes or selective removal of abundant proteins

8. 干扰物质 Proteases Nucleic acids Polysaccharides Plant phenols Lipids
Salt ions
Insoluble material

9. 如何使蛋白酶失活
Problem: Cell lysis → endogenous proteases set free → proteolytic attack → artifacts (Mr, pI)
Remedies:
protease inhibitors (2-D Protease Inhibitor Mix)
precipitation reaction (2-D Clean Up Kit)
boiling with SDS buffer

10. 常用的蛋白酶抑制剂 PMSF inhibits serin- and thiol-proteases
- is not stable in aqueous solutions
- is inactivated by DTT, ß-mercaptoethanol etc.
- is toxic (alternative: Pefablock™)
AEBSF (Pefabloc™) alters pI of some proteins
EDTA inhibits metallo-proteases
Pepstatin inhibits acidic proteases
high pH inhibits/slows down actions of proteases
Limits:
Protease inhibitors do not inactivated all proteases!
Proteins may become modified by protease inhibitors
According to Sigma:
AEBSF is directly soluble in water. Sigma tests the solubility of AEBSF at 50 g/ml in water yielding a clear colorless solution.
Solutions in water are slightly acidic and retain inhibitory activity for up to six months when stored refrigerated. Solutions at pHs above 7 are less stable.1,2 Stock solutions should be stored at a pH less than 7. If a final pH of greater than 7 is required, the pH adjustment should be done shortly before use.
References

11. 核酸和多糖 Removal of nucleic acids: Removal of polysaccharides:
cause horizontal and vertical streaks in 2-D patterns
increase sample viscosity
clog pores of PAGE
may cause background smear in silver stained 2-D patterns
Removal of nucleic acids:
treatment with a protease-free DNAse/RNAse mixture
TCA/acetone precipitation of proteins and re-solubilization
adding a basic polyamine (e.g. spermine) and ultracentrifugation
Removal of polysaccharides:
ultracentrifugation

12. 除去核酸的方法
DNase I and RNase A are commonly used (add 0.1x vol of 1 mg/ml DNase I, 0.25 mg/ml RNase A in 50 mM MgCl2)
Nucleases will not work in 8 M urea
DNase I will show up on a 2-D map. (pI ~5, MW ~30 kDa)
Benzonase (both DNase and RNase activity) is also commonly used.
Sonication works very well!

13. Effect of DNase Treatment
E. coli extract on 7 cm pH 3-10 NL
+ DNase
- DNase

14. 质脂
May interact with membrane proteins, “consume” detergents and form insoluble precipitates
Remedies:
organic solvents (ethanol)
phenol
protein precipitation (e.g. TCA/acetone, 2-D Clean Up Kit)
Limits:
loss of proteins (some may not re-solubilize)

15. 盐离子
High concentrations of salt ions interfere with IEF and cause overheating, “empty lanes”, disturbed 2-D gel patterns
Desalting:
minidialysis, spin dialysis or gel filtration (proteins can get lost)
protein precipitation (Ettan 2D Cleanup Kit, TCA-acetone); resolubilization with lysis buffer
“in-gel desalting”: dilute the sample and apply a larger volume instead (rehydration loading preferably)
“electrophoretic desalting I”: remove salt ions by appropriate electrode papers in a gel-side up focusing device (Ettan IPGphor Manifold, Multiphor II)
soft sample entry: low voltage for several hours
Limits: possible loss of proteins

16. Effect of salt
E. coli extract pH 4-7
no salt
30 mM NaCl

17. De-salting techniques
Dialysis
Spin dialysis
Gel filtration
Precipitation/ resuspension
Slow
Detergents can concentrate with protein
Protein losses
Complicated, can cause losses
Small ions can interfere with IEF, so it’s often advisable to de-salt a sample prior to 2-D.
Dialysis is very effective, and results in minimal protein losses, but it requires large volumes of solution and is time consuming.
Spin dialysis is faster. Some losses can occur. It should be borne in mind that spin dialysis can concentrate detergents along with protein, since detergent micelles have a high effective MW.
Gel filtration can be used for de-salting, but protein losses are often unacceptable.
Precipitation and resuspension as discussed earlier leaves small molecules and salts behind. It complicates sample prep, and can result in selective losses.

18. Effect of dialysis Pre-dialysis sample Dialyzed sample pH pH 5 6 7.510 pH 5 6
7.5 10

19. Desalting by Low Voltage IEF
Bovine vitreous proteins
18 cm strip, 50 µA / strip, 20 °C
Running conditions after the “sample entry” phase:
300 V 60 min 3500 V 42,000 Vh
Ref.:
Görg A, Boguth G, Obermaier C, Posch A, Weiss W. Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): The state of the art and the controversy of vertical versus horizontal systems. Electrophoresis. 16 (1995)
150 V / 30 min V / 5 hrs

20. Chaotropes Urea - efficiently breaks hydrogen bonds
- typically used as 8 to 9.5 M solution
Thiourea
- weakly soluble in water (1 M), but in concentrated urea solutions
( M)
- typically 2 M thiourea and M urea
- superior solubilizing power, especially for nuclear and membrane
proteins

21. Extraction:Comparison Urea vs Urea/Thiourea
The sample is rat liver. An equal mass of liver was extracted with either 8 M urea or 7 M urea, 2 M thiourea. The strip is 3-10 NL. Staining was mass spec-compatible PlusOne.
In addition to the above, the extraction medium contained:
4% CHAPS, 0.5% Pharmalyte 3-10, 2 mM tributylphosphine 1 mM PMSF
The rehydration solution was the same, but no PMSF and 60 mM DTT instead of TBP.
Note: The vertical streak in the acidic part of the urea/thiourea gel is very typical for thiourea. It is most probably not an impurity of thiourea, but comes from partically focusing of the thiourea itself.
8 M urea
Rat liver
7 M urea / 2 M thiourea

22. SDS in 2-D Sample Prep
Best solubilizing agent known but not compatible with IEF unless diluted into an excess of another detergent
Inhibits proteolysis
Useful with lipid-rich samples
Limited to low sample load
Can disturb first dimension

23. CHAPS vs. Amidosulfobetaine-C12
Spinach thylakoid membranes
2% CHAPS*
2% ASB C-12*
kDa
212
170
116 76 pH 7 10 7 10
*Extraction/rehydration also contained 7 M urea, 2 M thiourea, 0.5% Pharmalyte 3-10, 60 mM DTT

24. Reductants DTT (dithiothreitol) DTE (dithioerythreitol)
2-mercaptoethanol
Tributylphosphine(TBP)
Triscarboxyethylphosphine
(TCEP)
triscyanoethylphosphine
most commonly used
interchangeable with DTT
required at high concentration, contains impurities, but may have solubilization benefits (?).
Poorly soluble, very hazardous
Good reductant, but negative charge makes it unsuitable for 1st dimension.
Uncharged, soluble, but efficacy as reductant is in doubt.
DTT is a far better reductant than TCEP. In fact, TCEP has been used to study partially reduced disulfides due the the inability of TCEP to fully reduce disulfides

25. Sample treatment is very important
Cleanup from contaminants
Dissolve complexes completely
Protein-protein
Protein-polysaccharides
Stop protein activities (protease, phosphatases)
Precipitation is the most efficient…..

26. Protein precipitation
Clean-up from lipids, nucleic acids, polysaccharides, polyphenols, salts
Concentration of proteins
Irreversible inhibition of proteases
Prevention and dissolution of complexes
For DIGE: removal of endogeneous peptides

27. Protein precipitation procedures
Ammonium sulfate Not efficient, de-salting necessary, (salting out); NOT recommended
TCA precipitation Can be hard to re-solubilize
Acetone and/or ethanol Leaves SDS behind, but many proteins not precipitated
TCA plus acetone (Damerval et al. 1986) More effective than either alone, good for basic proteins
Chloroform and methanol (Wessels and Flügge, 1984) Time consuming, large volumes necessary
Cleanup Kit (TCA, detergent, acetone) 1 ½ hours, very efficient, good recovery
Most 2-D sample prep simply involves breaking open the cell to be analyzed, solubilizing the proteins in a solution suitable for IEF and spinning out debris. This is generally preferable both because it is simplest and also because it has the least potential for losing something you may be interested in. There are cases where a protein precipitation step is desirable. Precipitation accomplishes two things. It can concentrate protein from a dilute source, and it can serve to purify proteins away from substances that may interfere with 2-D separation.
Salt precipitation will only precipitate some of the proteins in a sample - it can, however, be used as an enrichment step, if you’re only interested in certain proteins that can be selectively precipitated. Salt interferes with IEF, so it has to be dialyzed out following resuspension.
Trichloroacetic acid is an effective protein precipitant. It has to be removed from the protein pellet prior to resuspension by extraction with an organic solvent such as acetone. The pellet may not resolubilize easily. some proteins may not be precipitated.
Organic solvents such as ethanol or acetone can be effective protein precipitants. These agents have the advantage that they leave some interfering substances behind, like lipid or detergent.
TCA and acetone together are very effective and can precipitate proteins that won’t come down with either agent alone. Damerval C, DeVienne D, Zivy M, Thiellement H. Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling protein. Electrophoresis 7 (1986)
Wessel D, Flügge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138 (1984)

28. Effect of sample precipitation
Crude E. coli lysate
E. coli lysate precipitated
with TCA/acetone and
resuspended
The gel on the left is a crude lysate of E. coli prepared simple by sonicating the cells in the presence of 8 M urea, 2% CHAPS and centrifuging. The streakiness towards the anode is most likely the result of anionic impurities in the lysate - phospholipids, nucleotides, polysaccharides etc.
This streaking disappears in a sample that has been precipitated with 10% TCA, 80% acetone and resuspended in 8M urea, 2% CHAPS. Method based on:
Damerval C, DeVienne D, Zivy M, Thiellement H. Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling protein. Electrophoresis 7 (1986)
Note that the treatment does alter the protein profile significantly.

29. Protein solubilization
ideal procedure:
disruption of all non-covalently and S=S-bound protein complexes and aggregates into a solution
ideal buffer:
cleaves all S=S-bridges, ionic bonds, H-bonds and hydrophobic interactions under conditions compatible with IEF and without modifying proteins

30. 蛋白质的有效溶解常用试剂 Urea (8-9.8 M) , or 7 M urea / 2 M thiourea
Detergent (CHAPS,…)
Reductant (DTT, 2-mercaptoethanol)
Carrier ampholytes (0.8 % IPG buffer)
Sonication can help solubilization
Sample can be heated only prior to addition of urea
Full solubilization may require several hours.
Proteins have to be very effectively solubilized prior to application to IEF. This can be troublesome, particularly with membrane proteins.
Solubilizing agents have to be non-ionic or zwitterionic so as not to interfere with IEF.
Most proteins are soluble in high concentrations of urea. Urea also fully denatures the protein, unfolding it so all the charged groups are exposed to the solution. Similar compounds such as thiourea have been used as well, and in some cases, are even more effective solubilizers.
Detergents are also used to keep proteins in solution and prevent them from aggregating. Detergents used in IEF have to be either non-ionic or zwitterionic usually it’s either CHAPS (zwitterionic) Triton X-100 or NP-40 (two similar non-ionic detergents. Anionic detergents (SDS or sarkosyl) are more effective solubilizers, but are normally incompatible with IEF. They can still be used (and often are) as long as they are diluted into a solution containing at least an 8-fold excess of non-ionic or zwitterionic detergent prior to IEF. T. Rabilloud has tested nondetergent sulfobetains and new detergents (2% ASB 14 or 16, or C 80) for improved soulbization of membrane proteins.
A reductant (usually DTT) is necessary to reduce all disulfide linkages and prevent oxidative aggregation. Lately alternatives have been suggested: TBP (tributylphospine) [TBP not recommended for hydrophobic proteins!!] or TCEP (Tris-(Carboxyethyl)-phospine). The latter should work very well, also regarding mass spectrometry analysis after 2 D electrophoresis)
Sonication accelerates the often tedious process of solubilizing a pellet.
Heating a protein sample in the presence of urea results in carbamylation, a modification that alters the pI of a protein. Heat can be used to aid solubilization, but it must be applied prior to addition of urea. Heating in the presence of detergent is a common means of sample solubilization.
Use of urea/thiourea mixtures: Rabilloud, T., Adessi, C., Giraudel, A., and Lunardi, J. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18: , Alkylamidosulfobetaine detergents: Chevallet, M., Santoni, V., Poinas, A., Rouquie, D., Fuchs, A., Kieffer, S., Rossignol, M., Lunardi, J., Gerin, J., and Rabilloud, T. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19: , 1998.

31. 促进样品复溶的有效方法 Pellet must not become completely dry!
Pipette repeatedly lysis solution over the pellet (do not vortex!)
Rehydration can take several hours (or over RT (do not vortex!)
Carefully sonicate (avoid heating of sample)
Use PlusOne Molecular grinding kit
Freeze pellet with lysis solution at 20 °C
Use SDS solution (2 % SDS, hot) and then dilute with 9 M urea / 4 % CHAPS
Perhaps: Try alternative procedure (Wessel and Flügge)
Wessel D, Flügge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138 (1984)

32. 特殊类型的样品 细菌 酵母(或其他真菌类) 培养的细胞 植物样品
High nucleic acid/protein ratio. Nucleic acid removal techniques are often employed
Tough cell walls require vigorous disruption techniques. Protease activity is high. SDS is usually used.
Salt carry-over from growth medium or wash solution can be significant. Salt-free buffer/osmoticum should be used for washing (10 mM Tris / 250 mM sorbitol pH 7.0).
Dilute source of protein. Precipitation is usually employed. Protease activity is high. Reductants and inhibitors are used to prevent phenolic modification.

33. Effect of sample prep technique (Drosophila larva extract)
Homogenate precipitated with
80% acetone, 10% TCA.
Resuspended in 8 M urea, 4% CHAPS
Homogenized in 8 M urea,
4% CHAPS
Homogenized in 2% SDS
Heated at 95 ºC 3 min
I hope this slide gives an impression of how important sample prep is.
These are extracts of 3rd instar Drosophila larvae prepared three different ways.
The gels were loaded on the basis of a constant mass of source material rather than constant amounts of protein.
The extract shown on the left was prepared simply by homogenizing the larvae in 8 M urea, 4% CHAPS plus PMSF. This is a standard sample prep technique, but it gives less than satisfactory results in this case.
The extract in the middle was prepared by homogenizing the larvae in 2% SDS and subsequently heating the extract. Despite the presence of SDS, the 2-D separation gives good sharp spots across a wide mass and pI range.
The extract on the right was prepared by precipitating the proteins on the homogenate with TCA and acetone, and resuspending in urea and CHAPS, another commonly employed technique. The spots obtained are sharp, but the protein yield is relatively low. This is probably because the precipitated protein was not able to be fully resuspended.
First dimension is pH 3-10 L run on IPGphor in 8 M urea, 2% CHAPS, 0.5% IPG buffer, 65 mM DTT

34. New Sample Preparation Kits from APBiotech
PlusOne Molecular Grinding Kit
PlusOne 2-D Clean-up Kit
PlusOne 2-D Quant Kit
PlusOne Microdialysis Kit
PlusOne PAGE Clean-up Kit (not for 2-D electrophoresis)

35. 2 D Clean-Up Kit for first-dimension IEF
Acidic precipitation with detergent co-precipitant
Washing of pellets with addition of organic solvents
Resuspension of pellets in sample solution

36. 2 D Clean-Up Kit for first-dimension IEF
Rat liver extracted with 4 % SDS, 40 mM Tris base
Untreated
pH 4-7
pH 4-7
Treated with PlusOne 2-D Clean-Up Kit

37. Cell extract Treated with PlusOne 2-D Clean-Up Kit Untreated
Stasyk T, Hellman U, Souchelnytskyi S.
Optimizing sample preparation for 2-D electrophoresis.
Life Science News 9 (2001) 9-12.

38. PlusOne 2 D Quant Kit
Compatible with...

39. PlusOne Microdialysis Kit
3 h to overnight

40. Effect of dialysis Pre-dialysis sample Dialyzed sample pH pH 5 6 7.510 pH 5 6
7.5 10

41. Effect of dialysis E.coli extract undialyzed
dialyzed with PlusOne Mini Dialysis Kit
pH 3-10 NL
pH 3-10 NL

42. DeStreak Reagent & DeStreak Rehydration Solution

43. DeStreak 20 µg reduced mouse liver proteins applied anodic
to Immobiline DryStrips pH 6–11, 11cm. Reswelling solution
containing either DTT (A & B) or DeStreak (C & D).
A & C:10.6 kVh;
B & D: 25.3 kVh.

44. Comparison of two 2-D electrophoresis maps generated with and without DeStreak in the strip rehydration buffer.

45. Conclusions Reproducibility is enhanced ;
Reproducible spot pattern is generated with strips covering different pH ranges;
A more cathodic position is observed for the majority of spots;
the spot pattern is completely stable over time in the first dimension;
The 2nd dimension is unaffected enabling the application of conventional protein identification/characterization techniques.