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Two-dimensional electrophoresis 2-DE
參考書:Westermeier R and Naven T (2002) Proteomics in Practice. WILEY-VCH
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Identification of 2-D Separated Proteins
Biological Material
tissue cells body fluids
2-D gel protei n spot
2-D gel spot immobilized
on a membrane
N-terminal sequence
N-terminal sequence
sequence sequence
fractionate
d peptide peptide mass map
peptide mix
N-terminal sequence
mass of protein
amino acid compositio
n co-
electrophoresis
SDS-PAGE peptide mapping
Immuno- staining
Edman degradation
MALDI-MSAmino acid analysis Edman
degradation blotting
Concentration SDS-PAGE
cleavage
elution Edman
degradation elution Biological
pre-
fractionation Biochemical pre-fractionation
HPLC SDS-PAGE
PSD-MALDI-MS nano-ESI-
MS/MS
blotting
Edman
degradationPSD-MALDI-MS MS/MS ladder sequencing
carboxy- peptidase ESI-MS MALDI-MS
deblocking
•Nuclei
•mitochondria
•membranes
•cytosol
•lysosomes
•Precipitation
•liquid chromatography
Bio-rad
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從1975年2D system 建立,經由30年後,為何沒有被取代 1.便宜
2.同時間,同地點 3.高通量
2-Dimension Electrophoresis (2-DE) for Protein Separation
The core technology of proteomics is 2- DE: At present, there is no other technique which is capable of resolving thousands of proteinsin one separation procedure.
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Sample preparation buffer composition IPG strip rehydration 1
stdimension: IEF run IPG strip wash
Equilibration buffer I Equilibration buffer II 2
nddimension: SDS-PAGE Gel staining (SYPRO ruby) Imaging
Two dimensional Electrophoresis (2DE)
1
2 3 4
5 6
7 6
Challenges for 2-D Electrophoresis
pH gradient?
Detection limit for low abundant proteins?
Loading capacity?
Hydrophobic proteins?
High molecular weight proteins?
7
Time line of 2D
Sample preparation:
IPG strip rehydration:
IEF run:
SDS-PAGE:
Gel staining:
?
12-18 hrs 12 hrs 4-10 hrs 13 hrs Experiment: ????
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Major technique in proteomic research:
2-D electrophoresis (separation)
Digest to peptide fragment MS analysis
1. First dimension:
denaturing isoelectric focusing
separation according to the pI
2. Second dimension:
SDS electrophoresis (SDS- PAGE)
Separation according to the
Interested spot
MW
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Advantage and Challenge for 2DE
• Provides a hard-copy record
of separation
• Allows facile (容易)
quantitation
• Separation of up to 9000
different proteins
• Highly reproducible
• Gives info on Mw, pI and
post-trans modifications
• Inexpensive
• Limited pI range (4-8)
• Proteins >150 kD
not seen in 2D gels
• Difficult to see
membrane proteins (>30% of all proteins)
• Only detects high
abundance proteins (top 30% typically)
• Time consuming
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電泳技術最早是由Tiselius在1937 年所發表的。他將此技術命名為移 動界限電泳技術( Moving Boundary Electrophoresis)。他的 做法是將蛋白質混和物置於一充滿 緩衝溶劑的U 型管中,並在U 型管 的二側施加電位,這會使得不同蛋 白質因具有不同的電泳遷移速率 ( electrophoretic mobility )而分離 開來。
電泳之先趨
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電泳的改良
在1939 年時,Konig 利用浸泡過電解質液 的濾紙條當作電泳的 固體媒介物成功分離 蛇毒中的色素,這也 開啟了帶狀電泳 ( Zone
Electrophoresis )的 里程碑。此後有許多 不同的材質也紛紛被 應用在電泳的分離。
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1959 年,Raymond 及Weintraub 提出以聚 丙烯醯胺聚合物( polyacrylamide; PA )作為 電泳的媒介物。聚丙烯醯胺聚合物不會對蛋 白質產生吸附作用,因此可有效改善濾紙電 泳中因濾紙本身的羥基( -OH )吸附生化分子 而產生Tailing 的現象。此項技術也被命名 為polyacrylamide gel
electrophoresis(PAGE) 。1967 年在 Shapiro、Vinuela 及Maizel 等人發表了有 關SDS-膠體電泳的技術( Sodium Dodecyl Sulfate Electrophoresis )。由於SDS-膠體 電泳的分離結果可以提供各分析物的分子量 大小的訊息,因此是目前最常被用來分離複 雜生化樣品如蛋白質的分離技術。至於隨後 發展出來的二維(2-D)膠體電泳則是在1975 年由Farrell 和Klose所發明。
SDS-膠體電泳的技術
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15 16
17 18
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Native gel electrophoresis
polypeptides retain their higher-order structureand often retain
enzymatic activity
and interactionwith other polypeptides migration of proteins depends on many factors, including size, shape, and native charge.• native gels omit (刪去) the SDS and reducing agent (DTT)
• do not put SDS or DTT in the sample buffer
• do not heat the samples
• prepare the gel and tank buffer solutions without SDS.
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Principle of the preparation of 2DE sample
1.蛋白質彼此表現量差異極大,針對研究目標,需有所取捨
2.針對hydrophobic 蛋白質時,需選擇適當之溶解劑
3.預防蛋白質被水解(low temperature, protease inhibitor, denature..)
4.避免使用任何可能會改變蛋白質特性(分子量,帶電荷)之化學試劑
5.儘量去除可能干劑分析之因子(如DNA, salt, )
6.使用高純度,高品質之化學試劑
7.保存sample時應低於-86oC
8.完整之實驗流程
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Interfering substances for 2DE
¾Lipids (detergents)
¾Proteases (inhibitor cocktails: prokaryote? or eukaryote?)
¾Nucleic acids (ultracentrifugation, nucleases)
¾Polysaccharides (ultracentrifugation)
¾Salts (dialyse; B less than 20 mM)
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Composition of standard lysis (rehydrolation) buffer (for
isoelectric focusing; IEF)
1. 9M urea (or 7M urea + 2M thiourea)
2. 4% CHAPS 3. 1% DTT
4. 0.8% carrier ampholyte 5. 0.02% bromophenol blue.
6. Protease inhibitor
1
2
3
5
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2-DE are in denaturing condition
Three components must present in 2-DE denaturing condition (namely, in IEF lysis buffer); It usually neutral condition
1. Urea (often > 7M)
2. Reductant (DTT used most widely) 3. Non-ionic or zwitterionic detergent
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sample處理
50mM Tris-HCl buffer,pH 7.4 .
100 ml mimiQ+0.788g Tris-HCl
先以pH7.4 Tris-HCl清洗生物樣品(組織 或細胞)去除干擾物質其步驟如下:
• 將生物樣品浸泡於Tris-HCl
•以震盪器震盪約一分鐘
•離心(25℃ 2000g 15分鐘 )後去上層液 體
•再重複上述三步驟一次
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Protein extraction
最後將生物樣品溶解於 IEF 溶液中:
•Urea 之作用最主要是要使蛋白質變性, Thiourea可有效 溶解疏水性或是巨 大蛋白質。但是 Thiourea 及其不純物 會干擾 IEF 中 pH 3~5 區域的品質。一般是以 7M Urea 及 2M Thiourea 混合使用,但最佳濃度則隨樣品而有所差 異。
• CHAPS為電中性的界面活性劑用以溶解脂質雙 層膜,因 其為電中性故不會影響蛋白質電性可保蛋白質等電點不變。
• bromophenol blue是為了之後方便觀測電泳進行狀況而預 先加入的染劑。
100μl of 0.1%(1mg / 1ml)to 50ml ddH2O 0.002% bromophenol
blue
7.6g 2M Thiourea
2g 4% CHAPS
24g 7M Urea
IEF溶液
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sample
Lysis buffer
Extraction
washing
Rehydration buffer (IEF buffer)
Centrifuge
Vacuum dry
IEF
Salt ↓27
Urea → denature
1. To convert proteins into single
conformation
by disrupting 2ndand 3rd structure.2. To avoid protein-protein interaction.
3. No effect IEF condition.
4. The purity of urea is very critical: heating or impurities must be avoid, because these would cause carbamylationof the protein, resulting in artifactual spots.
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Urea-induced carbamylation of proteins
分解
Up 30 OC, Urea become isocyanate → interaction with protein → carbamylation
H2O + NCO-
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Carbamylation induced mass shift
*Note: A proton is lost from the amino group on the protein during carbamylation and thus the change in composition is NHCO.
- 43.00582
* NHCO Carbamylation
43.00582 171.10078
C7H13N3O2 Carbamyl
Lysine
0 128.09496
C6H12N2O Lysine
Delta Mass Residue
Monoisotopic Mass Residue
Compositi on Amino Acid
15+1+12+16=44 loss a proton = 43
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Thiourea
for very hydrophobic proteins only (such as membrane protein).
To keep hydrophobic proteins into solution.
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CHAPS
1. Zwitterionic
detergent
2. Non-ionic polyol mixtures (tritonX-100 and Nonidet NP- 40), it higher purity
3. Increase solubility of hydrophobic protein
4. Non-denaturing5. Able to disrupt nonspecific protein interactions
6. Electrically neutral
7. Easily removed by dialysis
32 A buffer for hydrophilic protein 每50ml
5μl / 1ml 0.5% Ampholyte(or IPG buffer)
10mg / 1ml or 25μl/1ml 65mM DTE or 200mM TBP
100μl of 0.1%(1mg / 1ml)to 50ml ddH2O 0.002% bromophenol blue
2g 4% CHAPS
24g 8M Urea
B buffer for hydrophobic protein每50ml
5μl / 1ml 0.5% Ampholyte(or IPG buffer)
10mg / 1ml or 25μl/1ml 65mM DTE or 200mM TBP
100μl of 0.1%(1mg / 1ml)to 50ml ddH2O 0.002% bromophenol blue
1g 2% Salfobetaine 3~10
7.6g 2M Thiourea
2g 4% CHAPS
24g 7M Urea
C buffer for hydrophobic protein每50ml
5μl / 1ml 0.5% Ampholyte(or IPG buffer)
10mg / 1ml or 25μl/1ml 65mM DTE or 200mM TBP
100μl of 0.1%(1mg / 1ml)to 50ml ddH2O 0.002% bromophenol blue
7.6g 2M Thiourea
2g 4% CHAPS
21g 7M Urea
2-D rehydration buffer
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Other detergents (less use for IEF)
1. Triton X-100
(not easily remove and interfering MS)
2. Nonidet NP-40
3. SB3-10
4. SDS
1
2
4 3
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Reductant (DTE or DTT)
1. To prevent different oxidation steps of proteins.
2. 2-mercaptoethanol should not be used because its
buffering effect above pH 8.3. Keratin contamination might from 2-mercaptoethanol.
4. DTT (dithiothreitol) or DTE (dithioerythritol) are used
widely.5. DTT and DTE ionized above pH8. They move toward anode during IEF in basic pH gradient. It leads to
horizontal streaking
at basic area.DTE
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horizontal streaking in 2-D gel
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Basic area
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Other reduction methods
2-mercaptoethanol : pH >8; interaction with keratin,and resulting MS analysis uncorrectly TBP (tributylphosphine): very unstable.
An alternative way to adequate and reproducible 2- DE patterns in basic area:
1. Addition of higher amount of DTT to the gel 2. Addition of more DTT to a cathodal paper strip.
陰極
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Interfering substances
¾ Lipids (detergents)
¾ Proteases (inhibitor cocktails: prokaryote? or eukaryote?
)
¾ Nucleic acids (ultracentrifugation, nucleases)
¾ Polysaccharides (ultracentrifugation)
¾ Salts (dialyse; B less than 20 mM)
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COOH
NH
2H
+COO
-R - C - H NH
2H
+R - C - H
COO
-NH
2R - C - H
酸性環境 中性環境 鹼性環境
+1 0 -1
pK1~ 2
pK2~ 9
等電點
5.5
Juang RH (2004) BCbasics
The basic principle of pI or IEF
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IEF (isoelectric focusing) 非常重要 1. pI; isoelectric point: 指蛋白質於特定pH值下該蛋白質的總
淨電荷為”零”時,稱此時的pH值為此蛋白質之等電點 2. 需去除二級或三級蛋白質結構
3. When pH < pI 時(在比較酸的環境) → protein is positive charge → move to cathode; pH > pI 時 (比較鹼的環境)→
protein is negative charge → move to anode
4. 早期技術,carrier ampholytes(兩性物質)為介質加上high voltage → pH gradient (利用電場加注於兩性物質產生pH 值不同)
5. Immobilized pH gradient (IPG) 優缺點:
1.漂移 2.再現性 3.鹽類容忍度高
4.較多蛋白質分離 40
The characteristic pH at which the net electric charge is zero is called the isoelectric point or isoelectric pH, designated pI.
For glycine, which has no ionizable group in its side
chain, the isoelectric point is simply the arithmetic mean
of the two pKa values:
Acid base
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Principle of IEF
1. Protein (+) move to cathode, and protein (-) move to Anode 2. Electric field → carrier ampholytes → from anode to cathode
pH gradient (increase)
3. Every protein has its pI, When pH < pI 時 → protein is positive charge → move to cathode
pH gradient condition:
1. Solution (acid and base) formation 2. Electric field → ampholytes
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環境酸鹼度影響蛋白質的淨電荷
+ Net Charge of a Protein Buffer pH
Isoelectric point, pI
-
3 4 5 6 7 8 9 10
0 -
Juang RH (2004) BCbasics
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Traditional IEF procedure:
IEF in run in thin polyacrylamide gel rods (棒) in glass or plastic tubes.
Carrier ampholytes
Gel rods containing: 1. urea, 2. detergent, 3. reductant, and 4. carrier ampholytes (form pH gradient).
Need add electric buffer (long time induced interaction) Problem: 1. long time. 2. not reproducible.
In the past In the past
Ampholyte
Ampholyte
: 一個分子上同時帶有正電及負電基團 44Pharmalytes Ampholines
decreasing pI
electric field
long IEF time
where R = H or - (CH ) - COOH, x = 2 or 3
Pharmacia公司
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sample pH 9 -
pH 3 +
Isoelectric focusing (1stdimension)
General principle and protocol of 2-Dimension Electrophoresis
MW
pH gradient
SDS-PAGE Ampholytes
polyacrylamide
2nd dimension
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When pH < pI 時 → protein is positive charge → move to cathode;
pH > pI 時→ protein is negative charge → move to anode
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Traditional Equipment for Isoelectric focusing (IEF):
Ampholytes polyacrylamide
Cathode (-) electrode solution
Anode (+) electrode solution
48 sample
gel rod rebuffered in SDS buffer
Principle according to P.H. O arrell and J. Klose (1975) pH 10
pH 10
pH 3
pH 3 Isoelectric
Focusing in presence of urea, Nonidet NP-40 in vertical gel rod
First Dimension: Second Dimension:
SDS Polyacrylamide Gel Electrophoresis in discontinuous gradient gel
Separation acc. to Isoelectric Points (charge)
Separation acc. to Molecular Weight (mass)
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Ampholytes (up to 2%)
¾ Help protein solubilisation
¾ Scavenge cyanate ions (carbamylation↓)
¾ Precipitate nucleic acids (during centrifugation)
¾ Prevent interaction immobilines/protein
¾ Should represent the pH range desired
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Problems with traditional 1stdimension IEF
1.
Takes longer timeto run (may be renature).2.
Techniques are cumbersome (笨重). (the soft, thin, long gel rods needs excellent experiment technique)3.
Batch to batch variationof carrier ampholytes.4.
Patterns are not reproducible enough.5.
Lost of most basic proteins and some acidic protein.6.
Native protein good; denaturing not good51
Problems with the Carrier Ampholyte IEF
Batch to batch
reproducibility of the carrier ampholytes is inadequate(不合格)Carrier ampholytes gradients are unstable Limited protein loading capacity
Gradient drift causes lack of reproducibility
Gradient drift causes loss of basic and acidic proteins Soft gel rods have poor size stability
Personal skill
influences results52
The disadvantage of Traditional 2-Dimensional Electrophoresis
Anode (+) electrode solution Cathode (-) electrode solution
cathodic drift (陰極漂移)
Ampholyte polyacrylamide
pH 3 pH 3 pH 3 pH 9 pH 7 pH 5
Time
Add electrode solution → long time → interaction
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Resolution for IEF: Immobilized pH gradients (IPG)
gel film
1. The pH gradient is fixed, not affected by sample composition.
2. Reproducible data are presented.
3. Modified by Angelika Gorg by using thin film to support the thin polyacrylamide IEF gel, named Strips. (1988, Electrophoresis, vol 9, p 531)
Developed by Bjellqvist (1982, Biochem. Biophys Methods, vol 6, p317) PH gradient are prepared by co-polymerizing acrylamide monomers with
acrylamide derivatives (Immobilines) containing carboxylic and tertiary amino groups.
Immobilines are weak acid or weak base
CH2 CN C N
R = amino or carboxylic groups
H H O
CH2 CN C N
H R O
Acrylamide
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immobilized pH gradient ; IPG
IPG: immobilized pH gradients.
They do not disturb IEF like buffer addition, because they become uncharged when migrating to their pI.
1. To generate pH gradients
2. To substituting ionic buffer
3. To improve the solubility of protein
4. Dedicated (專注於) pH intervals, prepared for the addition to immobilized pH gradients, are called IPG buffer.
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Traditional method
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57 Gradient maker
plastic support film
Production of Immobilized pH Gradient (IPG) strip
A
C B
F E
acidic basic D
pH 3
pH 10 58
59 60
Zoom gels: narrow range
pH 3-6 pH 5-8 pH 7-10
pH 3-10
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Zoom gels: micro range
pH 4 pH 7
pH 4.7 pH 5.9
245 Spots
479 Spots
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IPG strip
Advantages:
• mechanically strong
• pH gradient cannot drift
• load larger amount of sample (dehydrated strip)
Disadvantages:
• membrane/hydrophobic proteins poorly represented on 2D
• some larger proteins lost (size exclusion)
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Dyes
1. To visualize the sample solution
2. To monitor the 2-DE running condition.
3. Bromophenol blue is low amount used do not disturb the analysis.
+
+ --
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Sample preparation
Cell or tissue Lysis solution Sonication vacuum Lysis solution Centrifugation
Measurement of [protein]
2-DE sample
66
1. Some proteases are also active in presence of urea and detergent.
2. PMSF is frequently used (8mM), toxic and short half-life.
3. Pefabloc (AEBSF) can also be used but modified proteins.
4. NO complete insurance against protease activity
5. Boiling sample in SDS buffer for a few seconds can inactive protease.
6. Precipitate proteins with TCA/acetone at -20C might inactivation protease activity.
Protease inhibitors
67
IPG strip rehydration and sample loading
2-DE sample Rehydration
solution Rehydration solution:
8M Urea 2% CHAPS
2% IPG buffer (Ampholyte) 0.28% DTT
Trace Bromophenol blue IPG strip holder
Position the IPG strip
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IPG strip rehydration and sample loading
Strip holder
Cathode (-) electrode
Anode (+) electrode
30 voltage 12hr
69
Critical issues in 2D gels
Reproducibility : multi-step procedure - open to variability Reliability (可信賴): particularly of quantitative data Validation (確認): use additional, complementary method
Sample preparation:
thorough, consistent method resulting in complete
solubilisation,disaggregation,denaturationand reduction of all proteins under electrophoresis conditions
cell disruption, protease activities, oxidation
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Measure the protein conc. in your samples.
1. Biuret
2. Lowry methods.
3. Bradford methods.
4. UV methods.
5. Special methods
6. Other commercial methods.
1. BCA assay (bicinchoninic acid assay, Pierce) 2. DC protein assay (detergent compatible, Bio-rad) 3. DC/RC protein assay (detergent/reducing agent
compatible, Bio-rad)
Before runninng IEF, you know the concentration of sample
Note: some interaction influence protein analysis
71 破碎細胞
溶於IEF溶液 的生物樣本
超音波振盪
獲得細胞破 碎之混合液
離心 上層液為溶有
蛋白質之溶液
下層為細胞 殘 骸
72 蛋白質的還原反應
還原劑(Reductants):
完成細胞破碎取得含蛋白質溶液後 即加入還原劑以進行反應破壞蛋白 質分子內之雙硫鍵(s-s) 。
早期是以 2-mercaptoethanol 來 還原樣品。目前大都於 dithiothreitol (DTT) 及 dithioerythritol (DTE)。因 為他們具有高純度及可在低濃度下 使用等特性(濃度一般為 20mM 到 100mM)。最近 tributylphosphine (TBP) 應用於提高疏水性蛋白質溶解 度,但由於 TBP 的不溶性與不穩定 性造成在 IEF 過程中相對無效來維 持蛋白質的還原狀態。
73
Ampholytes
利用此類試劑能維持樣 品溶解時 pH,並且能降低 電荷交互作用產生的聚集作 用。一般在樣品製備時,
ampholytes 濃度可達 2%
(v/v) 。
現在一般的用法都是使 用商業配方的Ampholyte,
它會搭配等電點預鑄膠條 IPG strip pH 梯度一起使 用,一般依所選用的IPG strip pH 梯度不同會選用該 規格專用之Ampholyte 。
Ampholyte IPG buffer
74
IPG strip ranges
IPG strips (3 mm x 18 cm x 0.5 mm)
¾ Narrow range
¾ Medium range
¾ Broad range
4 7
3.5 4.5 5.5 6.7
4.0 5.0 6.0
3 10
6 11
75
Broad pH range
(pH 3-10)
116 97 81 66
55 45
30
21
14 kDa
pI
3 4 5 6 7 8 9 10
76
Medium pH range
(pH 4-7)
116 97 81 66
55 45
30
21
14 kDa
pI
4 5 6 7
77
Narrow pH range (1 pH unit)
5.5 6.0
5.0 4.5
4.0
116
66 97
55 81
30 45
21
14
pI
MW (kDa)
(4.5-5.5)
(4.0-5.0) (5.0-6.0)
78
Run 2-DE, step by step
79
Run 2-DE step by step
80 等電點電泳儀
IPG strip
Holder
樣品點入凹槽內
等電點電泳儀
•首先在holder內點入上一單 元所述處理完成之樣品。
• 將IPG strip 覆蓋在樣品上,
使樣品溶液充分被IPG strip 吸收。
•取礦物油覆蓋在最上層,因 為樣品為水性溶液礦物油會浮 於上層(油水分層)會對樣品有 擠壓的效果曾加IPG strip 的 吸收效果,此外礦物油隔絕 樣品與空氣接觸可避免蒸 發。
•將holder置於等電點電泳儀之 電極板上
上層覆蓋礦物油 以防樣品蒸散
Holder cover IPG strip Electrode
Electrode pads
81
Loading for IEF In gel loading:
1.Usually use 2. Simple
3. Sample loading higher
Note:
1.long time (>12hr for rehydrolation) 2.Basic
SAMPLE
OIL
82
Highly Abundant Proteins
• Standard Strip Holder
• Cup-loading Strip Holder / Multiphor
Paraffin oil Paraffin oil
83
Cup loading:
1. Narrow range IPG 2. BASIC range
3. Must rehydration, then sample Note:
1.Lower sample loading 2.Slow movement 3.precipitation
Loading for IEF
Usually use:
1.Purified protein 2.High in glycoprotein 3.Very Basic protein (7-10)
4.High level of DNA/RNA or other large molecules in sample 5.Serum sample
Conventional and Universal Strip Holders
cup-loading stripholders
standard stripholders
85 參數設定
40000Vhr(時間╳小時) 8000V(伏特)
Step7
1 小時 6000V(伏特) Step6
30分鐘 3000V(伏特) Step5
30分鐘 2000V(伏特) Step4
30分鐘 1500V(伏特) Step3
30分鐘 1000V(伏特) Step2
30 分鐘 500V(伏特) Step1
等電點聚交 Temp = 20oC 12小時 30V
覆水(吸收樣品 ) Temp = 20oC 50 mA/strip
•首先設定IPG strip 覆水(吸收樣品 ) 所需電壓電流及作用時間
• 全程作用溫度均維持攝氏20度,以 避免urea在高溫時將蛋白質修飾而影 響等電點分離
• 全程作用溫度亦不可低於攝氏15度以 免urea結晶析出
• 等電點聚焦時先從低電壓開始,目 的是要將小分子的塩類及可能存在的 干擾物先行移至兩端之電極
• 之後逐漸增加電壓將蛋白質由小而大 逐一分離聚焦
86
IPGphor (IEF System)
Amersham Pharmacia Biotech Inc.
Protein IEF Cell
Bio-Rad Laboratories
Equipment for Isoelectric focusing (IEF):
87
Immobilized pH gradient strips (IPG strips)
Introduced by Introduced by GorgGorg. A. . A.
Ref: Ref: GorgGorg. A (1994), . A (1994), WestermeierWestermeier (2001)
(2001)
Dried gel strips can be stored at Dried gel strips can be stored at --20 20 to
to --80 from months to years.80 from months to years.
88
2-DE instruments, 1st dimension
Amersham Biosciences Bio-Rad
89
Staining of IPG Strips (cont. urea, detergent)
Acid Violet 17 Staining:
(Patestos NP et al. Electrophoresis. 9 (1988) 488-496)
• fix for 20 min in 20% TCA,
• wash for 1 min in 3% phosphoric acid,
• stain for 10 min in 0.1 % Acid Violet 17 solution in 10%
phosphoric acid,
• destain 3 ´ in 3% phosphoric acid until background is clear,
• wash 3 ´ 1 min with H2Odist,
• impregnate with 5 % glycerol,
• air dry.
90
1
stdimension – what to avoid
NaCl < 10 mM SDS < 0.25%
Tris < 50 mM phosphates nucleic acids lipids phenolics insoluble material heating
91
Special cases
Bacteria -high nucleic acid:protein ratio -use nucleic acid removaltechniques
Yeast/fungi -tough cell walls require vigorous disruption to lyse -protease activity high
Cultured cells -salt (especially phosphate ions) from medium -wash in salt free buffer / osmoticum
Plant tissues -dilute source of protein -precipitation is usually used -protease activity is high
-reductants/inhibitors to prevent phenolic modification
92
Focusing Time
Over Focusing
Under Focusing
93
After IEF run
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
94
第二次還原反應與甲基化 IPG strip
•待等電點聚焦完成後取出IPG strip ,以 二次去離子水沖掉礦物油並將IPG strip 轉 放至新容器
• 蛋白質之雙硫鍵於樣品處理步驟時雖已加 入還原劑打斷,但經過一段時間仍有再自 行產生鍵結的機會,因此再將IPG strip浸 泡於含有還原劑( dithiothreitol (DTT)、
dithioerythritol (DTE) 、 tributylphosphine (TBP)等….. )之平衡溶液中再行還原並與 SDS 反應
• 之後再將IPG strip浸泡於含IAA (Iodoacetamide)之平衡溶液中,行甲基化 反應 (alkylation) 固定還原的 –SH 為 –S- alkyl group ,如此可確保雙硫鍵不再形 成,此一甲基化反應不可用於樣品處理步 驟因為會影響等電點。
2 g 2 % SDS
30 g 30 % Glycerol
36 g 6 M Urea
3.3 ml 50mM 1.5M Tris pH=8.8
equilibrium buffer (平衡溶液)最終體積加 水至 100ml
95
Equilibration Buffer 1 (reduction) (10 ml/strip)
• 6 M urea
• 130 mM
DTT
• 30% glycerol
• 1.6% SDS
• 0.002% bromophenol blue
• 45 mM Tris base
• pH 7.0 (acetic acid)
(R-S-S-R’ x R-SH + R’-SH)
Strip equilibration-I
¾ 15 min rocking (room temperature)
¾ Pour off EB1
96
Strip equilibration-II
Equilibration Buffer 2 (alkylation) (10 ml/strip)
• 6 M urea
• 135 mM
iodoacetamide
• 30% glycerol
• 1.6% SDS
• 0.002% bromophenol blue
• 45 mM Tris base
• pH 7.0 (acetic acid)
(R-SH x R-S-CH
2-CO-NH
2)
¾ 15 min rocking (room temperature)
¾ Pour off EB2
97
DTT destroy disulfides bond IAA alkylated cys
98
Bis-Tris gels
Tricine gels
Tris-Glycine gels
Linear gels and gradient gels
SDS-PAGE
sodium docdecyl sulphate - polyacrylamide gel electrophoresis
99
• Only “Proteomics” is the large-scale screeningof the proteins of a sample (cell, organism or biological fluid), a process which requires stringently(嚴格) controlled steps of sample preparation, 2-D electrophoresis, image
detection and analysis, spot identification, and database searches.
• The core technologyof proteomics is 2-DE
• At present, there is no other technique that is capable of simultaneously resolving thousands of proteinsin one separation procedure and one time.
2D-electrophoesis gel (2-DGE)
100
This “O’Farrell” techniques has been used for 20 years without major modification.
20 x 20 cm (sometime 18 x 18) have become a standard for 2-DE.
Assumption: 100 bands can be resolved by 20 cm long 1- DE.
Therefore, 20 x 20 cm gel can resolved 100 x 100 = 10,000 proteins, in theory.
Evolution of 2-DE methodology
100 100 100 100
101
Why not using native condition
1. Under native condition, a great part of proteins exists in
several conformations. Different conformations →
induced different move in electric filed. This leads to more complex2-DE patterns.2. Native protein complexes sometimes too bigto enter the gel.
3. Reduction of protein-protein interactions.
4. For match the theoretical pI and MW, all proteins should not have 3D structure or quanternary structure.
102
103
β-mercaptoethanol
蛋白質 鏈狀 (peptide)
Sodium Dodecyl Sulphate (SDS)-陰離子界面活性劑
•SDS:Protein = 1.4:1 (結合重量比)
•單位質量蛋白質的陰電性均相同(charge/mass ratio) Î 單位質量蛋白質於電泳時所受電場的引力均相同
104
1. SDS
2. β- mercaptoethanol
(還原劑,使蛋白質之雙硫鍵打斷) 3. bromophenol blue (小分子指示劑) 4. glycerol
Sample buffer
SDS-PAGE Sample之處理
s s
SH SH
沸水浴中5分鐘
Sample buffer
取15 μl sample + 15 μl sample bufferBUT IN IPG strip did not need
如果是2-D的話要改成DTT or DTE
105
SDS
In IEF condition:Protein + Urea, DTT/DTE → denature protein
No IEF analysis:
protein
(heat 95oC)
No heat
SDS in running buffer
106
Acrylamide vs. Bis-acrylamide
Polyacrylamide) (%) moléculaire (KDa 15-20---10-40 10-15 ---40-100 5-10 ---100-300 5 ---300-500 2-5 ---PM>500
107
1. Acrylamide (A) :polymeration 2. Bisacrylamide (B) :cross-linkage
3. Ammonium persulphate (APS) :free radical donor 4. Tetramethylenediamine (TEMED) :attack APS
O
∥
(A)
CH2=CH–C–NH2
O O
∥ ∥ (B)
CH2=CH–C–NH–CH2–NH–C–CH=CH2
CONH2 CONH2 CONH︱
︱ ︱ ︱
–CH2–CH–CH2–CH–CH2–CH–CH2–CH–CH–
︱CONH
︱CH2
CONH CONH︱ 2 CONH2
︱ ︱ ︱
–CH2–CH–CH2–CH–CH2–CH–CH2–CH–CH–
︱CONH
︱
APS, TEMED
108
孔洞之大小(pore size)決定於
( 1)acrylamide與bisacrylamide總量
(2)bisacrylamide用量 ( 架橋程度 )
% T = total monomer concentration acrylamide + bisacrylamide
total volume X 100%
109 110 M.W.
KDa
111
gradient gels
112
113 114
Commercial gel
115
1. individual charge differences of the proteins are masked
2. hydrogen bonds are cleaved
3. hydrophobic interactions are canceled 4. aggregation of the proteins is prevented
5. removal of the secondary structure and ellipsoids are formed
SDS functions
116
117
Principle of electrophoresis
sample
Stacking
Separation
Protein + SDS → protein (negative charge)
-
+ 台大莊榮輝教授
Glycine pI=6.9
glycine Cl
-Gly
buffer中含glycine,沒有氯離子
膠體中的緩衝液 含氯離子,沒有 glycine
兩極之間一定 要有負離子來 帶動電流
118
•Running buffer:Tris-HCl , glycine pH 8.3
•Stacking gel: (pH 6.7)
H
3N
+CH
2COO
-+ H
+Cl
-> Gly
-> protein
-•Separating gel: (pH 8.9)
H
3N
+CH
2COO
-+ H
+H
3NCH
2COOH
Cl
-> protein
-> Gly H
3NCH
2COOH
Stacking gel
Discontinous Gel Electrophoresis
119
焦集膠體的[焦集作用]及其作用機理:
(1) 樣本分子所以能被焦集成一薄層,請注意下面三種分子在電泳時的表現:
(a) Glycine:圖中以黑點(當環境pH>6.9時,帶負電之glycine)或白點(當環境 pH=6.9時,不帶電之Zwitter ion)表示。
(b) 樣本分子,以P表示(蛋白質)。
(c) 氯離子,以斜線部分代表。
(2) 請注意上述電泳的五個部分,膠體中的緩衝液含氯離子,沒有glycine;然而tank buffer中含glycine,沒有氯離子。
(3) 再看[樣本溶液-焦集膠體-分離膠體]三段的pH是不連續的,其pH分別為[8.3-6.9- 8.9],注意glycine的pI恰為6.9。
(4) 當電泳一開始時,glycine一越入焦集膠體後,立刻變成不帶電的分子(白點),泳 動率很小;同時氯離子很快的往正極泳動,因此在氯離子與glycine之有一段缺乏 離子的空間,電壓很高。
(5) 然而兩極之間一定要有負離子來帶動電流,此時只有利用蛋白質分子來傳導, 而焦集膠體中的孔隙又較疏,於是蛋白質分子在此[離子缺乏空間]快速往正極泳 動,一直碰到氯離子的尾端,而聚集於斯,成一薄層,由側面觀之則成一細線。
(6) Glycine分子慢慢通過焦集膠體,又成為負離子,[離子缺乏空間]瓦解;樣本蛋白 質泳動到分離膠體中,膠體為正常使用濃度,開始依其分子量,電荷等因素泳動。
Typed by Hua 120
Separating gel 12 % Acrylamide (Tris-HCl, pH8.9)
Stacking gel 3.5% Acrylamide ( Tris-HCl, pH6.7)
Running buffer : Tris - HCl, glycine, pH 8.3
Discontinous Gel Electrophoresis
121
1-D gel 2-D gel
122
SDS Gel Electrophoresis (agarose or polyacrulamide)
Denaturing condition
Denature protein by adding SDS (then separate by size only)
Used to estimate purity and molecular weight, separate proteins by size Electrophoresis of SDS-solvated protein on polyacrylamide gel Stain gel with Coomassie Blue (binds to proteins)
SDS forms micelles and binds to proteins
123
2-D gel electrophoresis equipment - 2nd dimension
various lengths linear / gradient reducing / non-reducing
Multi-gel runners = higher reproducibility
HoeferDalt Multiple Slab Gel Unit
Casting of up to 23 gels 10 SDS gels
125
SDS PAGE in Ettan DALT II
126
2-DE instruments, 2nd dimension
16 x 16 cm 8 x 10 cm
23 x 20 cm
Amersham Biosciences
127
2-DE instruments, 2nd dimension Bio-Rad
128
Traditional method- tris glycine gel
tricine
129
After IEF → SDS-PAGE
130
Run 2-DE, a quick overview
131 第二維電泳分子量分離
• 將處理完畢之IPG strip轉移 到SDS PAGE 上並注入0.5%
的agarose黏合
• 將電泳槽一一組裝完成注入 上層電泳液
• 調整適當電流後啟動
132 第二維電泳分子量分離
• 將處理完畢之IPG strip轉移 到SDS PAGE 上並注入0.5%
的agarose黏合
• 將電泳槽一一組裝完成注入 上層電泳液
• 調整適當電流後啟動
133
蛋白質體學 蛋白質體學
研究流程 研究流程
134
從生物樣本中萃取蛋白質 從生物樣本中萃取蛋白質
萃取 萃取 與 與 分離 分離
蛋白質 蛋白質
135
將蛋白質溶液點入電泳溝槽 將蛋白質溶液點入電泳溝槽
等電點
等電點(IEF)(IEF)分離分離 136
覆蓋等電點預鑄膠片條 覆蓋等電點預鑄膠片條
等電點 等電點(IEF)(IEF)分離分離
+ -
等電點 137
等電點(IEF)(IEF)分離分離
+ -
等電點預鑄膠片條復水 等電點預鑄膠片條復水
138
等電點電泳分離 等電點電泳分離
等電點等電點(IEF)(IEF)分離分離
139
將等電點膠片條轉移至垂直電泳槽 將等電點膠片條轉移至垂直電泳槽
140
將等電點膠片條轉移至垂直電泳槽
將等電點膠片條轉移至垂直電泳槽
141
將等電點膠片條轉移至垂直電泳槽 將等電點膠片條轉移至垂直電泳槽
142
將等電點膠片條轉移至垂直電泳槽 將等電點膠片條轉移至垂直電泳槽
143
分子量垂直電泳分離 分子量垂直電泳分離
144
分子量垂直電泳分離
分子量垂直電泳分離
145
分子量垂直電泳分離 分子量垂直電泳分離
146
分分 子 子 量 量 分分 離 離
分子量垂直電泳分離 分子量垂直電泳分離
147
分 分 子 子 量量 分 分 離離
分子量垂直電泳分離 分子量垂直電泳分離
148
Protein detection (staining) in 2-DE gel
good UV or laser normal
simple 1~10 ng SYPRO Ruby Stain
bad normal special
complex 0.6~1.2 ng Silver Stain
good normal normal
simple 8~28 ng Coomassie Stain
reproducibility Image
scan Before MS analysis operation
sensitivity stain
Coomassie blue
Simple, reproducible, reasonable dynamic range but not very sensitive
Silver stain
Multiple steps, variable dynamic range but sensitive, MS compatibility
Sypro ruby
Simple, good dynamic range, sensitive but expresive
Other methods
Isotope, immunodetection,…
149
Staining protocol
• Fixative (30 min)
•
SYPRO ruby (12 hrs)
• Washing (30 min)
• 2% glycerol storage solution
• Store gels at 4°C
(40% Methanol - 10% acetic acid)
(10% Methanol - 6% acetic acid)
Protect from light
150
Imaging
• UV detection (300 nm)
• Blue light (470 nm) t 5 min B Fuji Imager
4 pI 7
5 6
kDa 116 97 81 66
55 45
30
21
14
MW
151
主要染色法比較
• 一般常見的染色法共計三種Coomassie stain 、Silver stain 、 Fluorescent stain(螢光染色) 。
•Coomassie stain是目前最簡單也最經濟的染色法,其最大缺點就是敏感 度差微量蛋白質不易被染出或者是蛋白質表現量相近時不容易辨別其差 異。
•Silver stain是敏感度最佳的染色方式,但它的致命缺點就是於顯影步驟 時其顯影時間難以被掌握,往往可能一兩秒內就造成很大的顏色深淺差 異,又常因個人認知之主觀差異常使顯影時間過長而不自知,因此很難從 顯影條件不一致的膠片得到準確的相對定量分析 。
•Fluorescent stain(螢光染色)顧名思義就是利用螢光化合物對膠體內的蛋 白質染色,由於目前商品化產品種類眾多,我們今天只選擇最廣泛使用的 SYPRO Ruby 介紹。
•SYPRO Ruby Stain之敏感度雖不及Silver stain ,但也足以應付目前蛋 白質體研究的主要需求,其使用方式簡便沒有過度顯影的問題每次染色品 質一致,其最大缺點就是價格較高,且必須使用特別的影像系統例如:UV box 或昂貴的雷射掃瞄器等….. 。
152
Sensitivity limit
Quantification Living cells Linear Dynamic Range Coomassie Blue
staining
100 ng +++ no 3
Negative staining 15 ng + no 3
Silver staining 200 pg ++ no 7
Fluorescent staining
400 pg ++++ no 104
Fluorescent labelling
250 pg ++ no 104
Radioactive labeling:
X-ray film 1 pg +++ yes 20
Phospor-imager plates
0.2 pg ++++ yes 105
Stable isotope labelling
< 1 pg ++++
(with MS)
yes ?
153
• Only high-resolution 2-DE with both dimensions run under denaturing conditions is used.
• Native 2-DE plays no big role.
• Goal: to separate and display all gene products present.
Today 2-DE
154 SYPRO Ruby染色法
• 固定:以7% 醋酸和10% 甲 醇 浸泡凝膠約三十分鐘後倒 掉,再加入二次去離子水清 洗一次
•加入Sypro Ruby染色至少三 小時。
•退染:再以7% 醋酸和10%
甲醇 浸泡凝膠約三十分鐘後 倒掉,再加入二次去離子水 清洗一次。
•由於Sypro Ruby會與玻璃反 應,為了避免Sypro Ruby與 儀器上的玻璃反應所以一定 要將凝膠表面之Sypro Ruby 洗去。
Critical Issues of 2-D Electrophoresis
• Sample preparation and sample application
• Loading capacity
• Protein transfer 1st − 2nd dimension
• Multistep procedure
• Reproducibility of spot positions
Sample Preparation
• Cell disruption
• Protein precipitation
• Solubilization
• Protection against protease activities
• Removal of
– nucleic acids – lipids
– salts, buffers, ionic small molecules
– insoluble material
Factors to consider
• 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
Cell disruption methods
• Freeze-thaw or osmotic lysis
• Detergent lysis
• Sonication
• Enzymatic lysis
• French pressure cell
• Grinding (mortar and pestle)
• Mechanical homogenization
159
Nucleic acid removal
• 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 MgCl
2)
• 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!
Effect of DNase Treatment E. coli extract on 7 cm pH 3-10 NL
+ DNase - DNase
Protein precipitation
• Ammonium sulfate (salting out)
• TCA precipitation
• Acetone and/or ethanol
• TCA plus acetone
• Not efficient, de-salting necessary
• Can be hard to re- solubilize
• Leaves SDS behind, but many proteins not precipitated
• More effective than either alone, good for basic proteins
Effect of sample precipitation
Crude E. coli lysate E. coli lysate precipitated
with TCA/acetone and resuspended
Protein solubilization
• 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
Extraction:Comparison Urea vs Urea/Thiourea
7 M urea / 2 M thiourea
Rat liver
8 M urea
165
Reductants
• DTT (dithiothreitol)
• DTE (dithioerythreitol)
• 2-mercaptoethanol
• tributylphosphine
• triscarboxyethylphosphine
• 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.
166
Protease inhibitors
• PMSF
(phenylmethyl sulfonyl fluoride)
• AEBSF (Pefabloc)
• EDTA
• Peptide protease
inhibitors
(leupeptin aprotinin etc.)
• High pH
• Most commonly used
Inactivates serine and cysteine proteases
Is inactivated by DTT and 2- mercaptoethanol
• More soluble, less toxic than PMSF, but can cause charge modifications(?).
• Inhibits metalloproteases
• May show up in 2-D pattern
• Inhibits most proteases, but avoid Tris base
167
Effect of salt
E. coli extract pH 4-7
no salt 30 mM NaCl
De-salting techniques
• Dialysis
• Spin dialysis
• Gel filtration
• Precipitation/
resuspension
• Slow
• Detergents can concentrate with protein
• Protein losses
• Complicated, can
cause losses
169
Effect of dialysis
Pre-dialysis sample Dialyzed sample
pH5 6 7.5 10 pH5 6 7.5 10
170
Desalting by Low Voltage IEF
150 V / 30 min 100 V / 5 hrs
Bovine vitreous proteins
171
Special cases
• Bacteria
• Yeast (and other fungi)
• Cultured cells
• Plant tissue
• 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 / 25 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.
Effect of sample prep technique (Drosophila larva extract)
Homogenized in 8 M urea, 4% CHAPS
First dimension is pH 3-10 L run on IPGphor in 8 M urea, 2% CHAPS, 0.5% IPG buffer, 65 mM DTT
Homogenized in 2% SDS Heated at 95 ºC 3 min
Homogenate precipitated with 80% acetone, 10% TCA.
Resuspended in 8 M urea, 4% CHAPS
173
Challenges for 2-DE
1. Spot number:
10,000-150,000 gene products in a cell. Usually it is impossible to display all proteins in a 2-DE gel.
PTM makes it difficult to predict real number.
Sensitivity and dynamic range of 2-DE must be adequate.
174
Challenges for 2-DE
2. Isoelectric point spectrum:
– pI of proteins: range from pH 3-13. (by in vitro translated ORF)
– PTM would alter the pI outside this range.
– pH gradient from 3-13 dose not easy exist.
– For proteins which pI > 11.5, they need to be hand separately.
175
Challenges for 2-DE
3. molecular weights:
Small proteins or peptides can be analyzed by modifying the gel and buffer condition of SDS-PAGE.
Protein > 250 kDa do not enter 2ndSDS-PAGE properly.
176
Challenges for 2-DE
4. hydrophobic proteins:
Some very hydrophobic proteins do not go in solution. Although thiourea…..x
Some hydrophobic proteins are lost during sample preparation and IEF.
More chemical developments are required.
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Challenges for 2-DE
5. Sensitivity of detection:
Low expressed proteins are very difficult to detect, even employing most sensitive staining methods.
Increase sensitivity, induced noise.
Sensitivity of staining methods:
1. Silver staining 2. Fluorescent staining 3. Dye binding staining (CBR)
178
Challenges for 2-DE
6. Loading capacity:
For detection of low abundant proteins, more sample needs to be loaded.
A wide dynamic range of the SDS-PAGE is required to prevent merging of highly abundant protein.
Loading capacity: IEF > SDS-PAGE.
179
Challenges for 2-DE
7. Quantitation:
The detection method must give reliable quantitative information.
Silver staining does not give reliable quantitative data.
180
Challenges for 2-DE
8. Reproducibility:
Highest importance in 2-DE experiment.
Immobilized pH gradient strip have improved a lot for 1
stdimension consistency
Variation most comes from sample preparation.
181
Time
Sample preparation:
IPG strip rehydration:
IEF run:
SDS-PAGE:
Gel staining:
Total: ~ 4 days 2-3 hrs 22 hrs 24 hrs 19 hrs 13 hrs
Experiment and sample collection182
2-DE needed major progress
Sample preparation and solubilization
Insoluble protein: such as membrane proteins Protein form highly resistant tissue like hair and skin
Low abundance proteins Prefraction
High sample loading Basic proteins Quantitation
183 184
For very hydrophobic proteins
Membrane proteins do not easily go into solution. A lot of optimization work is required.
1. Thiourea procedure 2. SDS procedure
3. New zwitterionic detergent and sulfobetains
185
Thiourea vs. hydrophobic protein
1.
7M urea + 2M thiourea (Rabilloud, 1998) → dissolve ↑2.
Increase spot number considerably.3.
Causing artifact spots.4.
Causing vertical streaking at acidic area.Lysis buffer, 8M urea Lysis buffer, 7M urea+ 2M thiourea 186
2-DE advantage and defect
Advantage:
1.Resolve a relatively large number of proteins 2.Economical
Defect:
1.Sample impurity effect resolution
2. Limited solubility of hydrophobic proteins
3.Difficulty in focusing highly basic and acidic protein 4.Time
5.Sensitivity, low abound protein not easy analysis 6.Limited MW range (>180 kDa)
7.Automation not easy
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Central tool for proteomics
Developed in 1975 (O’Farrell and Klose)
Requirement: sample from different subjects (control and test; treated and non test; ); Several gels and high repetitive
Computer analysis
Statistical tool: variation up to 40% between a gels of a same sample → produce 3-5 gels from identical material for better results
2-DE in proteomics
188
Main approaches to 2-DE analysis
Creation of proteome maps: systematic analysis of all spots on a gel---- one gel, and all proteins
Monitoring of gene product over a set of 2-DE gels--- set of gels, selected proteins
Differential expression of proteins: global investigation between 2-DE image of several populations---several gels, differential analysis