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1

Protein

separation, purification and analysis

2

■ Sigma 目錄的試劑標籤:

品名 純度

批號 目錄編號

包裝

警告

警告符號 防護

貯藏

別名 分子式 含水量

CAS 編號

條碼

Sigma Catalogue

3

■ 常見的警告符號:

爆裂物 氧化劑 易燃物 毒藥

刺激性 腐蝕性 生物傷害 環境傷害

4

Properties of proteins

Complex: macromolecules Amino acids

Joined together by peptide bonds Precise length made in a linear

Many are modified (changes size, changes charge, function) Distinctive 3D shape

(2)

5

Protein Mass and concentration

1. Protein mass is measured in Daltons (Da)/ kDa 2. One Dalton = 1/12 the mass of a 12C atom

3. Average, the molecular weight of amino acid is 100 Da (1kDa)

4. Most proteins range from 30-80 kDa

5. Trp and Try have a high ability to absorb light with maxium absorption at 280 nm. Since most proteins contain these amino acids, protein concentration can be estimated spectrophotometrically.

6

Proteins are the molecule tools for most cellular functions

TYPE FUNCTION EXAMPLE Structural proteins Support Collagen, Elastin,

Keratin Storage proteins Storage of amino acid Ovalbumin,

Casein Transport proteins Transport of other

substrate

Hemoglobin

Hormonal proteins Coordination of and organism’s activities

Insulin

Receptors proteins Response of cell to chemical stimuli

Receptor in nerve transmit route Contractile proteins Movement Actin, Myosin Defensive proteins Protecton against

disease

Antibodys

Enzymatic proteins Selective acceleraton of chemical reactions

Trypsin, ATPase, GAPDH Signal transduction Signal regulated MAPK

7

Possible sources of protein

Outside a cell (extracellular)

-milk, secretion, lymph, plasma….

Inside a cell (intracellular)

-cytoplasma, nuclear extract Part of a cell (organelle)

-membrane, nucleus, golgi, mitochondria…..

8

Why is Localization Important?

• 1974 Nobel Prize in Physiology/Medicine

– George Palade

• “for discoveries concerning the structural and functional organization of the cell”

• 1999 Nobel Prize in Physiology/Medicine

– Günter Blobel

• “for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell”

細胞的核糖體是蛋白質合成的工廠

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9

The differences between Protein and DNA

Size

Solubility (H2O)

Concentration

Cleaving enzyme

Isoelectric point

Residues

Define wide

Highly highly-poorly

Even 1012

About 100 few

4.5 2-13

4 20 DNA Protein

10

Sample preparation is most important step in proteomics

1. Good sample preparation is the key to good proteomics 2. Minimum to avoid sample loss

3. Minimized the proteolytic degradation 4. Reproducible method

5. Remove the interference 6. Avoid sample contamination 7. Avoid co-analytical modification 8. Decrease temperature

9. High selective procedure for cell/tissue analysis (tissue specific, Laser capture micro dissection)

General consideration

11

Some important concepts for sample preparation 1. A good sample preparation is the key to good result.

2. The protein composition of the cell lysate or tissue must be reflected in the patterns of 2-DE.

3. Avoid protein contamination from environment.

4. Co-analytical modification (CAM) must be avoided. (pre- purification sometimes leads to CAM)

5. Highly selective procedure for tissue analysis (Laser capture micro dissection, LCM)

6. Treatment of sample must be kept to a minimum to avoid sample loss.

7. Keep sample as cold as possible.

8. Shorten processing time as short as possible.

9. Removal of salts

10. Minimized the unwanted processing, eg proteolytic 12

¾Laser capture microdissection (LMD or LCM)

1. A slide with sectioned tissue is placed under the microscope.

2. The cell or cell cluster of interest is selected by the user using the computer mouse.

3. The laser cuts out the cell(s).

4. The captured cell(s) are collected in an eppendorf tube.

5. Cells are ready for molecular analysis.

Principle:

small areas – even single cells- are cut out of tissue sections with a laser

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13

Microdissection Strategy

UV Cutting & Gravity

LMD Laser Micro-Dissection

( Leica AS LMD Laser Microdissection System)

14

Methodology

Epp gravity

Glass slide

Plastic foil

Tissue

Laser

15

Laser Capture Microdissection

16

Sample (protein) preparation 1. Tissue or cell sample

2. Protein solubilization 3. Purification?

4. Is need Protein precipitation?

5. Inhibition of protease activity 6. Removal of non-protein

lipids

salts, buffer, ionic small molecules nucleic acids

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17

Why needs pure proteins

Aim amount required purity Crystallography 100 mg high >95%

Enzyme kinetic 10 mg medium-high Antibody production 1 mg medium-high Therapeutic g-kg 99.9%

Biophysical 1-10 mg 95-99 % Multiprotein study 1-10 ug high

Mutagensis study 1-2 mg medium-high

18 Various protease inhibitors

在研究蛋白質的相關技術中,最重要的是預防蛋白質被分解

19

Nucleic acid removal

1. Benzonase (both DNase and RNase activity; endonuclease ) most

commonly used

2. DNase I and RNase A are commonly used

3. 8 M urea (denature protein include nuclease)

4. Sonicationworks (mechanical disruption)

Nucleic acids macro molecular, interaction with many protein Possible method

20

Cell lysis (disruption) methods 1. Sonication

2. Freeze-thaw (nitrogen) 3. Osmotic lysis

4. Pressure cell 5. Enzymatic lysis 6. Detergent lysis

7. Other Mechanical homogenization

AVOID 1. High temperature 2. Contamination 3. Long Time 4. Oxidation

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21 22

Waring blender

23

Animal cell and plant cell

24

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25 26

Freeze-thaw (nitrogen)

Nitrogen

27

Mechanical homogenization

Polytron

28

Can not disrupted some cell organs

To separation of mitochondira, nucleus….. protein Tissue grinders

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29 30

31

French Press

法式高壓破碎機

樣本加壓快速通過Sealed plug與Release valve之間的細孔,以剪力破壞細胞;一般 建議細胞體以20%~30%的比例與懸浮緩衝液充分混合後並於上機前充分排出樣本室 內的空氣便可以達到最佳的打破效果。和常用的Sonicator相比較,French Press可

以一次處理更大量的樣本,並更方便於全程保持低溫以確保樣本品質。 32

A cross-section through the Manton-Gaulin homogeniser valve, showing the flow of material. The cell suspension is pumped at high pressure through the valve impinging on it and the impact ring. The shape of the exit nozzle from the valve seat varies between models and appears to be a critical determinant of the homogenisation efficiency.

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33 34

Homogenization of solid sample Bead beater

35 36

球磨機 (ball mill)

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37

機械式粉碎機

38

39

Separation of nucleus protein form cell or tissue

Cell /tissue + isotonic buffer →Dounce grinder (homogenizer) → stroke → microscope observation (trypan blue) → centrifuge 900 g → pallet

Wash with isotonic buffer

↓900g

twice

Nuclear

Add hypotonic buffer

Centrifuge

Supernatant (nuclear)

Supernatant (cytosol, synaptosome)

40

Centrifugation

A centrifuge is used to separate particles or even macromolecules:

-Cells

-Sub-cellular components -Proteins

-Nucleic acids Basis of separation:

-Size -Shape -Density Methodology:

-Utilizes density difference between the particles/macromolecules and the medium in which these are dispersed

-Dispersed (被驅散) systems are subjected to artificially induced gravitational fields

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41

Sedimenation coefficient

Relative centrifugal force (RCF)=1.12r(rpm/1000)2

Principle of centrifugation

Suspension In process

Induced gravitational field

Precipitate Supernatant

Separation complete

42

RCF or g force and RPM

• Relative Centrifugal Force (RCF)

– The force during centrifugation that moves the particles outward from the center of rotation – Also know as the g force

• Revolutions per minute (rpm)

– The number of times the a rotor completes a revolution in one minute of centrifugation – The RCF and rpm are related but dependent

on the characteristics of the rotor (the radius)

43

Calculating g force from rpm

Relative centrifugal force (RCF)=1.12r(rpm/1000)2 RCF = 11.2r(rpm ÷ 1000)2

RCF = 11.2 × 25.4 × (4200 ÷ 1000)2= 5018g

已知一個離心機轉頭的半徑r=254mm (25.4cm), 速度rpm=4200, 求 RCF?

44

dr/dt d2(ρp − ρm)ϖ2r

S = V/ϖ2 r = ─── (V= ──────)

ϖ2 r 18η 沈降系數(S)

顆粒在單位離心力作用下的沈降速度稱該顆粒的沈降系數

沈降系數是生物大分子的特徵常數,它除了與顆粒的密度、形狀和 大小有關以外,還與介質的密度、黏度有關。

為了紀念Svedberg人們把沈降系數確定為S,單位是︰1S=10-13秒。

如︰某物質沈降系數是10-12秒,就可寫成10×10-13秒,表示為10S.

Sedimenation

Sedimenationcoefficientcoefficient

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45

Ribosomes

Origin Complete

ribosome Ribosomal

subunit rRNA

components Proteins Cytosol

(eukaryotic ribosome)

80 S 40 S

60 S

18 S 5 S 5.8 S 25 S

C.30 C.50

Chloroplasts (prokaryotic ribosome)

70 S 30 S

50 S

16 S 4.5 S

5 S 23 S

C. 24 C. 35

Mitochondrion (prokaryotic ribosome)

78 S ∼30 S

∼50 S

18 S 5 S 26 S

C. 33 C. 35

46

離心機的分類與構造 離心機的分類︰

離心機有一系列的商品。它沒有一個統一的分類標準或規定,但 大家公認的分類辦法有兩種︰

按離心速度或離心力分類︰

低速離心機︰最大轉速(Vmax)為 2000-6000 rpm 高速離心機︰最大轉速(Vmax)為 6000-25000 rpm 超速離心機︰最大轉速(Vmax)為 40000-100000 rpm

Centrifuge 離心機

47

角型(angle rotor):典型的離心方式,多用在大量製備時。

懸籃式(swing bucket rotor):傳統用在密度梯度離心或細胞。

垂直(vertical rotor)

The types of rotor

48 Rotor

Centrifuge tube

Supernatant Precipitate

Suspension

swing bucket rotor

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49 50

51 52

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53

Separation of organelles of by density-

grandient centrigugation

Subcellular fractionation

54

Size → differential centrifugation

Larger objects will precipitate at lower speeds C & F FA.1

55

Subcellular Fractionation from different centrifuge

tissue homogenate

1000 g

Pellet unbroken cells

nuclei chloroplast

transfer supernatant

transfer supernatant

transfer supernatant

10,000 g 100,000 g

Pellet mitochondria

Pellet microsomal

Fraction (ER, golgi, lysosomes, peroxisomes)

Super.

Cytosol, Soluble enzymes

56

Ultracentrifugation

•Rotates at high speeds e.g. 30000 rpm

•Ultracentrifuges:

•Analytical ultracentrifuge (AUC) is mainly used for studying properties of macromolecules

•Preparative ultracentrifuges are used to separate macromolecules such as proteins and nucleic acids

•The high speeds used in such devices generate considerable amounts of heat

•Therefore cooling arrangements are required in ultracentrifuges

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57

Isopycnic Equilibration (CsCl 梯度形成) Zone Centrifugation

(預鑄梯度) 高 速 離 心

Gravity Centrifugation (無梯度)

■ 高速離心與兩種超高速離心法的比較:

樣本:多為蛋白質 密度相似、分子量不同者

樣本:多為核酸 密度不同、分子量相似者 一般的重力離心僅把

顆粒與溶液分離開來

(Supercoiled) RNA Plasmid DNA (Open circular) Chromosomal

DNA Protein

Density Density

Density Density

Lipids

(階段梯度)

台大莊榮輝教授

58

Difficulties to isolation of membrane protein

Need triton X-100 to disrupt the membrane

59

Separate protein from membrane and cytosol

Spinal cord tissue homogenized Centrifuge (600g)

Nuclei supernatants centrifuge (100,000g) Pellet supernatants

lysis (tritonx-100)

cytosolic protein Centrifuge (100,000g)

Supernatants

Synaptosomal membrane protein

60

Difficulties to disrupt the plant cell wall

2nd metabolite may play important role of medicine

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61

Difficulties to the plant cell or tissue Cell wall: hard to disrupt

Chloroplast: pigment interaction with gel…

Vacuole: many special interference 蛋白酶 (proteases)

多酚化合物 (polyphenols):

Alkaloid 生物鹼 Flavonoid類黃酮

Tannin 單寧

Phenolic compound pigment β-mercaptoethanol

Phenol oxidase inhibition

Polyvinylpolypyrrolidone (PVPP)

adsorb

62

Detection of low abundance proteins-sample pre-fraction

1. Subcellular fractionation 2. Purification (Chromatography) 3. Electrophoresis

4. Differential solubilization (polar, non-polar solution) 5. Others

63

Separation of protein method

Many properties of proteins can be used to isolated proteins

Proteins which are similar in one property man differ in other properties

Property method

Solubility Ammonium sulfate precipitation Size dialysis, gel filtration

Charge IEF, ion exchange chromatography Affinity affinity chromatography

64

Table of common methods of protein purification

Property Methods

Size / shape Size-exclusion chromotography electrophoresis Isoelectricpoint

(charge)

Ion exhange chromatography IEF

binding to small molecules

Affinity, antibody chromatography solubility Precipitation with

ammonium sulfate (salting out)*

Purification procedures attempt to maintain the protein in native form. Although some proteins can be re-natured, most cannot!

To purify a protein from a mixture, biochemists exploit the ways that individual proteins differ from one another. They differ in:

– Thermal stability*

*For most protein purifications, all steps are carried out at ~5°C to slow down degradative processes.

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65

Protein precipitation

1. Concentrate protein samples.

2. Removal of several disturbing material at the same time.

3. Inhibition of protease activity.

1. Ammonium sulfate precipitation(Not efficient, de-salting necessary)

2. TCA precipitation(Can be hard to resolubilize)

3. Acetone (ethanol)precipitation (Leaves SDS behind, but many proteins not precipitated)

4. TCA/Acetoneprecipitation (More effective than either alone, good for basic protein)

Method

66

Ammonium sulfate precipitation

Proteins tend to aggregate in high concentration of salt (salting out)

• Add Ammonium sulfate slowly into solution and stir for 10-30 mins

• Harvest protein by centrifugation.

Limitation

• Some proteins are soluble at high salt conc.

• Ammonium sulfate seriously affect IEF.

67

TCA precipitation

Trichloroacetic acid is a very affective protein precipitant.

• Add TCA to extract to final conc.10-20%.

• Add 10-20% TCA directly to tissue or cells.

• Harvest protein by centrifugation.

• Wash access TCA by ethanol or acetone.

Limitation

• Sometimes the pellet is hard to redissolve.

• TCA must remove complete. (affecting IEF)

• Some degradation or modification of protein occurs

68

Acetone precipitation

The most common organic solvent used to precipitated proteins, lipid and detergent remain in solution.

Add at least 3 vol. of ice-cold acetone into extract.

Stand on ice for at least 2 hours.

Harvest protein by centrifugation.

Remove access acetone by air drying.

Limitation

Sometimes the pellet is hard to redissolve.

Some proteins would not precipitate.

DNA/RNA and glycan also precipitate.

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69

Example, Acetone precipitation

With Acetone precipitation Crude extract by lysis buffer

70

TCA/acetone precipitation

The method is more active than TCA or acetone alone. Most commonly used in 2-DE.

Suspension samples in 10% TCA/Acetone with 0.07%

2-mercaptoethanol or 20mM DTT.

Stand on -20C for at least 45mins.

Harvest protein by centrifugation.

Wash the pellet by acetone with0.07% 2- mercaptoethanol or 20mM DTT.

Remove access acetone by air dry.

Limitation

Sometimes the pellet is hard to redissolve.

TCA must remove complete. (affecting IEF)

Some degradation or modification of protein occurs

71

Precipitation with ammonium acetate in methanol following phenol extraction

The method is more suitable for plant sample with high level of interfering substance

Proteins are extracted into buffer saturated phenol.

Precipitated by ammonium acetate/methanol.

Harvest protein by centrifugation.

Wash with ammonium acetate/methanol followed by acetone.

Limitation

Complicated.

Time consuming.

72

C D

A B

Σ 823 spots Σ 757 spots

Σ 969 spots Σ 899 spots

Acetone Isopropanol

TCA TCA/Acetone

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73

Differential solubilization

74

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

75

Thiourea procedure

7M urea + 2M thiourea (Rabilloud, 1998) good: Increase spot number considerably.

bad: Causing artifact spots.

Causing vertical streaking at acidic area.

76

Example, thiourea procedure

Lysis buffer, 8M urea Lysis buffer, 7M urea+ 2M thiourea

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77

SDS procedure

For emergency case.

Up to 2% SDS can be used.

Have to dilute SDS samples at least 20 fold with urea an a non or zwitterionic detergent containing solutions.

The major reasons for using SDS:

1. Formation of oligomers can be prevented 2. Dissolved tough cell walls samples (with

boiling)

3. Dissolved very hydrophobic proteins

78

New zwitterionic detegent and sulfobetains

Three major types of detergent

1. Non ionic detergent

Triton x-100, Tween 20, Brij-35 2. Ionic detergent

SDS, CTAB, Digitonin 3. Zwittergent

CHAPS, CHAPSO, Zwittergent 3-08, 3-10, 3-12…

79

層析

80

Column Chromatographyfor separation of protein

Liquid flow

Liquid flow

4:37

Time 1 2 3 4 5

Separation according to:

-molecular weight/ size -charge

-hydrophobicity -affinity

Sample containing proteins or peptides

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81 82

Fraction Collector

83 84

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85 86

Gel filtration chromatography

87

Bead (有孔小珠)

88

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89

Ion-exchange chromatography

90

Separation by binding affinity

Affinity chromatography

The natural binding activity (or antibody specific characterization) of a protein can be used to purify it.

By attaching its ligand to a complex mixture through the column and only your desired protein will be retained

It can be released by add free ligand with competes for protein sites Examples:

– antibody columns - monoclonal antibody attached to column – glutathione column – binds glutathione-S-transferase on

target

91 92

Technique End conditions Start conditions

Small sample volume GF Diluted sample

Buffer change (if required)

Low ionic strength IEX High ionic strength or

pH change

High ionic strength HIC Low ionic strength

Specific binding conditions AC Specific elution conditions

Linking Chromatography Techniques

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93

fast protein liquid chromatography (FPLC)

94

95 96

High Performance Liquid Chromatography (HPLC)

column

Detector

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97 98

99 100

Size → dialysis

• Use membranes (plastic, cellulose) with holes of a defined size to separate molecules based on their physical size

– Small molecules can pass through small holes

• At equilibrium concentrations of molecules that can pass through the membrane will be the same on both sides

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101 102

微膜離心管: 離心管中橫置一微膜,利用離心力把小分子 擠過,大分子留在上方;樣本

數目多而體積少時,多採用此法。實驗室較常用的是 Stirred cell(Amicon), Centricon(Centriprep)。

103

Salting in / Salting out

• Salting IN 鹽溶

• At low concentrations, added salt usually increases the solubilityof charged macromolecules because the salt screens out charge-charge interactions.

• So low [salt] prevents aggregation.

• Salting OUT 鹽析

• At high concentrations added salt lowers the solubility of

macromoleculesbecause it competesfor the solvent (H2O) needed to solvate the

macromolecules.

• So high [salt] removes the solvation sphere from the protein molecules and they come out of solution.

104

Salting IN 鹽溶

Salting OUT 鹽析

Protein surface (hydorphobic)

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105 106

107 108

Electrophoresis Purification (Chromatography)

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109

Protein sample preparation, purification and separation

What needs to preparation, purification and separation of protein?

What preparation, purification and separation techniques used in proteomics?

To remove abundant proteins To enrich low abundant proteins To remove non-protein interaction To reduced the protein complexity To extract the interesting proteins

Depended on what research target are interested

Based on the diverse properties of protein

110

How to keep the protein activity ? 1. Fast

2. Low temperature 3. Inhibition of protease 4. Buffer solution 5. Contamination

111 112

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113

Purification is a multi step procedure (Intro – flow chart. )

Sample Separation

technique

Fractionation

Purification is a multi-step procedure.

Is there activity?

Set aside

No Combine

Fractions

yes Monitor

purity

Assay total protein Assay enzyme activity

Pure?

Prepare for analytical technique yes

No

Repeat with another separation technique until pure

114

純化步驟設計

高活性:protein的比活性要能顯著提高,各種protein因材料 來源及含量多寡不一,純化倍率也有高低;不過就同一 樣本而言,當然越高越好。

高回收率:一般指總活性的回收。

高純度:純度與活性是protein純化的兩大目的,以達到均質 protein為最終目標;相對而言,在電泳上看不到其它雜 質,即可視為均質。

方便與快速:方法要儘量簡便,步驟勿拖太久,因為酵素活 性可能隨著時間而急速降低

經濟:許多試劑相當昂貴,大量使用時要考慮錢的問題。

115

Protein from cells or tissue

First steps: Preparing the sample – Crude extract.

Microbial cells or tissue

Break cells, tissue, or organ Blender, homogenizer, sonication, pressure, psmotic

Pellet with intact cells, organelles, membranes and membrane proteins

Supernatant with Soluble protein

116

Collect fractions.

• Fractionation

• Text:

As the column separates the proteins in the mixture, the

“effluent” drips into a series of fraction tubes that are moving at a specific rate of speed.

These tubes are called fractions.

Here we are showing 20 tubes.

Fraction collectors in most labs have about 75-200 tubes.

Be sure to remove color from column as it drips into the tubes below! If the sample is spread over three tubes, the center tube will be darker in color.

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117

• Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer.

• The values can be plotted against the fraction number in is what is called an elution profile.

• Notice the peaks on the graph.

These indicate where the fractions are that contain protein.

Question 1. How do we know which fractions contain protein?

A280 0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction

#

A280

Fraction # Peaks

118

• Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein.

• Notice the results of the enzyme assay.

The highest activity corresponds to one of the peaks.

Question 2. Which fractions contained the desired enzyme?

A280 0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction

#

A280

Fraction # EnzAssay

Results

Need to substitute values for the colored spots since we are switching to an absorbance based assay.

Now we can have them discard tubes that don’t have enzyme activity.

119

Combine (pool) the fractions with activity.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction

#

Fraction #

Pool fractions

1. NEXT We want to pool the fractions that have enzyme activity.

It may be useful to consider more than just the activity of a fraction. Specific activity is a measure of the amount of enzyme activity per amount of protein (units/mg). The higher the specific activity, the higher the purity. When pooling fractions, judgement is needed as to whether to optimize yield or specific activity.

120

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121

Measure the protein conc. in your samples.

– Widely used protein assay methods 1. Biuret

2. Lowry methods.

3. Bradford methods.

4. UV methods.

5. 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)

122

123 124

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125 126

127 128

bad

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129

Biuret Test

Peptide Chains Biuret Complexes ( purple color )

– Principle: The reactivity of the peptide bonds with the copper [II]

ions under alkaline conditions to form purple biuret complex.

– Interfering substance: Ammonium sulfate, Tris, etc.

– Sensitivity: >mg

Requires large amounts protein (1-20mg)

Low sensitivity 130

Folin-Ciocalteu ( Lowry ) Assay

Lowry, OH, NJ Rosbrough, AL Farr, and RJ Randall. J. Biol. Chem. 193: 265. 1951.

Oostra, GM, NS Mathewson, and GN Catravas. Anal. Biochem. 89: 31. 1978.

Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

Hartree, EF. Anal Biochem 48: 422-427 (1972).

131 132

• Sensitive over a wide range

• Can be performed at room temperature

• 10-20 times more sensitive than UV detection

• Can be performed in a microplate format

• Many substances interfere with the assay (Strong acids, ammonium sulfate )

• Takes a considerable amount of time to perform

• The assay is photosensitive, so illumination during the assay must be kept consistent for all samples

• Amount of color varies with different proteins

Lowry Assay

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133

BioRad DC Protein Assay

• Based on Lowry Assay with following improvements:

1. Reaches 90% of its maximum color development within 15 minutes

2. The color changes not more than 10% in 2 hours

134

135

Bicinchoninic Acid ( BCA ) Assay

136

Very sensitive and rapid if you use elevated temperatures

Compatible with many detergents Working reagent is stable

Very little variation in response between different proteins

Broad linear working range

The reaction does not go to completion when performed at room temperature

Bicinchoninic Acid ( BCA ) Assay

(35)

137

Dye-Binding ( Bradford ) Assay

Bradford, MM. A rapid and sensitive for the quantitation of microgram

quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.

Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

CBBG primarily responds to arginine residues (eight times as much as the other listed residues)

If you have an arginine rich protein, You may need to find a standard that is arginine rich as well.

CBBG binds to these residues in the anionic form Absorbance maximum at 595 nm (blue)

The free dye in solution is in the cationic form, Absorbance maximum at 470 nm (red).

138

Dye-Binding ( Bradford ) Assay

• Fast and inexpensive

• Highly specific for protein

• Very sensitive [1-20 µg (micro assay) 20-200 µg (macro assay)]

• Compatible with a wide range of substances

• Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin)

• Dye reagent is complex is stable for approximately one hour

• Non-linear standard curve over wide ranges

• Response to different proteins can vary widely, choice of standard is very important

139 140

1.Protein sample 2.Kit Buffer

(36)

141

Enzyme-immuno assay reader (EIA-reader, ELISA-reader)

Enzyme-linked Immunosorbent Assays (ELISAs)

142

• Pipet 5 ul of standards and samples into a microtiter plate

• Add 25ul of reagent A into each well

• Add 200ul reagent B into each well

• Agitate the plate to mix the reagents

1 2 3 4 5 6 7

A 0 0.24 0.48 0.96 1.20 1.44 empty

B 0 0.24 0.48 0.96 1.20 1.44 empty

C 0 0.24 0.48 0.96 1.20 1.44 empty

D blank blank blank blank blank blank empty

E 0 0.1C 0.2C 0.3C 0.5C C empty

F 0 0.1C 0.2C 0.3C 0.5C C empty

G 0 0.1C 0.2C 0.3C 0.5C C empty

H empty empty empty empty empty empty empty Blank=Reagent A+BEmpty= Air 0=Reagent A+B+5ul water

143 144

BioRad DC Protein Assay

• After 15 minutes, absorbance can be read at 750nm.

The absorbance will be stable for about 1 hour

Standard Protein Sol .BSA (750nm)

0.45 0.47 0.49 0.51 0.53 0.55

0 20 40 60 80

Time(min)

Absorbance

From Oliver

(37)

145 Bradford分析法

Bradford蛋白質分析法原 理是利用Comoassie Brilliant G-250酸性溶液 與蛋白質鍵結後其最高吸 光度會從465nm轉到 595nm,其操作步驟是先 準備已知濃之蛋白質標準 品以分光光度計分別測量

其吸光度然後繪製標

準曲線再利用標準曲

線求出未知蛋白質樣品濃 度 。

蛋白質標準品 0.2mg/mg

蛋白質標準品 0.4mg/mg

蛋白質標準品 0.6mg/mg

蛋白質標準品 0.8mg/mg 未知濃度

蛋白質

Protein (mg)

A595(吸光度) 0.2mg

0.4mg 0.6mg 0.8mg

未知濃度 蛋白質

146

Detergent effect High BCA assay method, detergent effect low

147

Background high

148

(38)

149 150

Commercial methods (kit) for the concentration of protein

A.A.BCA assay (BCA assay (bicinchoninicbicinchoninicacid assay, Pierce)acid assay, Pierce)

B. DC protein assay (detergent compatible, Bio B. DC protein assay (detergent compatible, Bio--

rad) rad)

C. DC/RC protein assay (detergent/reducing C. DC/RC protein assay (detergent/reducing

agent compatible, Bio agent compatible, Bio--rad)rad) This process is a two-step reaction.

Protein + Cu2+ + OH- Cu1+

Cu1+ + 2 BCA Cu1+/BCA chromophore (562 nm).

151

Lowry Absorbance

280 nm Absorbance

205 nm Bradford Dye-binding

Method sensitivity

0.05 - 5 mg 0.05 - 0.5 mg

0.05 - 2 mg 0.01 - 0.05 mg 0.01 - 0.05 mg

accuracy

high Medium

low high Medium

呈色快速 有腐蝕性 銨離子干擾大

呈色較慢 多種離子有干擾

樣本可回收 其它物質干擾大

樣本可回收 氧分子干擾極大 呈色快 雜質干擾多

顏色不易洗除

Biuret

參考文獻

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