Cara Kerja Plasmin

8/10/2019 Cara Kerja Plasmin

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Plasmin is produced in thebloodto break downfibrin,the major constituent of bloodthrombi,

thereby dissolving clots once they have fulfilled their purpose of stopping bleeding. Extra

production of plasmin caused by streptokinase breaks down unwanted blood clots, for example,

in thelungs(pulmonary embolism). The usual activation of Plasminogen (Plgn) is

byproteolysisof the Arg561Val562 bond.[6]

The amino group of Val562 then forms a salt-bridge

with Asp740, which triggers a conformational change producing the active protease Plasmin(Pm). When (SK) is present, it binds to Plgn to form a complex (SK. Plgn) that converts substrate

Plgn to Pm. Residues 159 of SK regulate its capacity to induce an active site in bound Pg by a

nonproteolytic mechanism and to activatesubstratePg in a fibrin-independent manner. This

complex subsequently rearranges to an active complex although the Arg561Val562 bond

remains intact. Therefore anotherresiduemust substitute for the free amino group of Val562 and

provide a counterion for Asp740 in this active complex.[7]

Two candidates for this counterion have

been suggested: Ile1 of streptokinase and Lys698 of Plgn. Deletion of Ile1 of SK

markedlyinhibitsits capacity to induce an active site in plasminogen, which supports the

hypothesis that establishment of a salt bridge between Ile1 of SK and Asp740 of plasminogen is

necessary for SK to induce an active site in plasminogen by a nonproteolytic mechanism.[8]

In

contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation

constant of the SK complex by 15 to 50 fold. These observations suggest that Lys698 is involved

in formation of the initial SKPlgn complex.

The mechanism of human plasminogen (HPlg) activation by streptokinase (SK)-type activator was

investigated with recombinant truncated SK peptides. An enzyme-substrate intermediate of HPlg.SK.

HPlg ternary complex was demonstrated by a sandwich-binding experiment. Formation of the ternary

complex was saturable, HPlg-specific, and inhibited by 6-aminocaproic acid. Three interaction sites

between SK and HPlg were demonstrated. SK-(220-414) bound to HPlg with two binding sites: one to

the micro-HPlg region, the catalytic domain of HPlg, and one to the kringle 1-5 region, with Kd values

of 1.50 x 10(-7) and 2.44 x 10(-6) M, respectively. SK-(16-251) bound to a single site on the kringle 1-

5 region of HPlg with a Kd of 4.09 x 10(-7) M. SK-(220-414) and SK-(16-251) competed for binding onthe same or nearby location on the human kringle 1-5 domain. Combination of SK-(220-414) and SK-

(16-251), but not either peptide alone, could effectively activate HPlg. In addition, SK-(16-251) dose-

dependently enhanced the activation of HPlg by SK-(16-414), while the HPlg activation by SK-(16-

414) was inhibited by SK-(220-414). We conclude that the HPlg that binds to the COOH-terminal

domains of SK functions as an enzyme to catalyze the conversion of substrate HPlg that binds to the

NH2-terminal domain of SK to human plasmin.

Structural studies of the Human Plasminogen Catalytic DomainAuthors:

Rao,Ramya

Project Name:

Protein Structures - Midterm due 10/16/08

Introduction

A zymogen is an enzyme precursor that requires a modification to convert itself to its active form.

Usually, a hydrolysis or cleavage reaction makes the active site more accessible to the substrate.

The paper by Wang et al. discusses plasminogen, a zymogen whose activation by proteolytic

cleavage to plasmin plays a key role in fibrinolysis leading to dissolution of blood clots. Two

proteases, tissue-type plasminogen activator and urokinase are responsible for the activation.

Plasminogen consists of seven independently folded domains. A growth-factor like domain forms

the N-terminus, which is followed by five kringle domains and a C-terminal trypsin-like catalytic

domain. Fibrin binding sites are present in the kringle domains. When there is no fibrin, the
http://en.wikipedia.org/wiki/Bloodhttp://en.wikipedia.org/wiki/Bloodhttp://en.wikipedia.org/wiki/Bloodhttp://en.wikipedia.org/wiki/Fibrinhttp://en.wikipedia.org/wiki/Fibrinhttp://en.wikipedia.org/wiki/Fibrinhttp://en.wikipedia.org/wiki/Thrombushttp://en.wikipedia.org/wiki/Thrombushttp://en.wikipedia.org/wiki/Thrombushttp://en.wikipedia.org/wiki/Lungshttp://en.wikipedia.org/wiki/Lungshttp://en.wikipedia.org/wiki/Lungshttp://en.wikipedia.org/wiki/Proteolysishttp://en.wikipedia.org/wiki/Proteolysishttp://en.wikipedia.org/wiki/Proteolysishttp://en.wikipedia.org/wiki/Streptokinase#cite_note-pmid5643782-6http://en.wikipedia.org/wiki/Streptokinase#cite_note-pmid5643782-6http://en.wikipedia.org/wiki/Enzyme_substratehttp://en.wikipedia.org/wiki/Enzyme_substratehttp://en.wikipedia.org/wiki/Enzyme_substratehttp://en.wikipedia.org/wiki/Residue_(chemistry)http://en.wikipedia.org/wiki/Residue_(chemistry)http://en.wikipedia.org/wiki/Residue_(chemistry)http://en.wikipedia.org/wiki/Streptokinase#cite_note-pmid45675-7http://en.wikipedia.org/wiki/Streptokinase#cite_note-pmid45675-7http://en.wikipedia.org/wiki/Streptokinase#cite_note-pmid45675-7http://en.wikipedia.org/wiki/Enzyme_inhibitorhttp://en.wikipedia.org/wiki/Enzyme_inhibitorhttp://en.wikipedia.org/wiki/Enzyme_inhibitorhttp://en.wikipedia.org/wiki/Streptokinase#cite_note-PMID10213631-8http://en.wikipedia.org/wiki/Streptokinase#cite_note-PMID10213631-8http://en.wikipedia.org/wiki/Streptokinase#cite_note-PMID10213631-8http://en.wikipedia.org/wiki/Streptokinase#cite_note-PMID10213631-8http://en.wikipedia.org/wiki/Enzyme_inhibitorhttp://en.wikipedia.org/wiki/Streptokinase#cite_note-pmid45675-7http://en.wikipedia.org/wiki/Residue_(chemistry)http://en.wikipedia.org/wiki/Enzyme_substratehttp://en.wikipedia.org/wiki/Streptokinase#cite_note-pmid5643782-6http://en.wikipedia.org/wiki/Proteolysishttp://en.wikipedia.org/wiki/Lungshttp://en.wikipedia.org/wiki/Thrombushttp://en.wikipedia.org/wiki/Fibrinhttp://en.wikipedia.org/wiki/Blood


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molecule adopts a closed' conformation. When it binds fibrin, plasminogen changes to an open'

conformation, as a result of which it has a significantly higher activation rate. Located in the

catalytic domain of plasminogen is an activation loop in which an Arg561- Val 562 peptide bond is

cleaved upon activation of the zymogen. This cleavage leads to certain conformational changes,

such as the formation of a salt-bridge that is not accessible to solvents. Thus, the functional active

site is formed. The activation loop of plasminogen is unique because it is restrained by a disulfide

bond between Cys558 and Cys556. This restraint is the cause of unusual properties seen in

plasminogen activation.

Figure 2: Activation loop of plasminogen showing the disulfide bond between Cys558 and Cys559.

Picture produced using Chimera2

The authors set out to study the 3-D structure of the catalytic unit of plasminogen to understand

how various plasminogen activators activate this zymogen. Their work showed that certain loops in

human plasminogen undergo dramatic changes in conformation upon activation to plasmin. Thesechanges cause a reduction in the S1 specificity pocket in plasminogen relative to plasmin, and also

skew the location of the disulfide bond relative to chymotrypsinogen-chymotrypsin. It is important

to understand the structure of the catalytic site of plasminogen (Plg), as it will lead to the

development of new and improved thrombolytic, anti-cancer and anti-pathogenic drugs1.

Materials and Methods

The structure was determined by X-ray crystallography upto 2.0 resolution. A mutant form of

plasminogen was used, where the active site serine residue at position 741 was changed to an

alanine residue. This was done in order to prevent possible proteolysis.

Mutant plasminogen was expressed as inclusion bodies from pET11 vector, using the E. coli. strain

BL21 as a host. The protein was re-folded using established protocols and was passed through a


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Sephacryl S-300 sizing column, followed by an ion exchange column. Buffers contained 20 mM

HEPES pH 7.0, 400 mM urea, and an NaCl gradient from 0.0 to 1.0 M.

Crystal trays were set up using the screen MembfacTM, manufactured by Hampton Research.

Crystals were grown in the sitting drop method. The condition in which the crystals grew was

12%(w/v) PEG4000, 100mM NaCl, 10% (v/v) isopropanol and 100mM N-(2-acetamido)

iminodiacetic acid pH 6.5. The dimensions of the crystals were approximately 0.2 mm x 0.2 mm x

0.05 mm.

X-ray diffraction data was collected at Brookhaven National Labs with a 1.04 wavelength

synchrotron light source. Diffraction data sets were collected at 2.0 with a completeness of 82%.

The structure was refined to an R-factor of 19.4% and an Rfreeof 26.7%. There are four protein

molecules per asymmetric unit, and the solvent content is 49%. Molecular replacement using a

model of human Pm (catalytic site of plasmin) was performed using the program AMoRe, and

refinement was carried out using X-PLOR.

Results and Discussion

Plg contains 2 six and seven-stranded -barrels, each one of them forming the core of a

subdomain. The 2 domains, termed N and C domains and connected through 3 trans segments

and the A-chain peptide, which spans residues 542 though 557. Perpendicular to the junction of

the two subdomains sits a cleft in which the active site is located. There are several loops and 2 -

helices located on the surface of Plg. The 94-shunt, 60-loop, 37-loop and the calcium-binding

loop make up the N-domain. The C-domain consists of the methionine-loop, S1-entrance frame,

oxyanion-stabilizing loop, 186-loop and the autolysis loop. These loops surround the active site.

The autolysis loop contributes to the activation pocket structure. The loop is quite mobile, and this

mobility may be attributed to the formation of the salt bridge within the activation pocket when

plasminogen is proteolytically cleaved. This salt bridge is formed when, upon activation, the -

amide group of Val562 shifts by approximately 12 into the activation pocket and interacts with

Asp740. Due to the formation of the salt bridge bond, a zymogen triad consisting of Ser578,

His586 and Asp740 that stabilizes the plasminogen structure gets disrupted upon activation to

plasmin. The oxyanion-stabilizing loop and the S1 specificity pocket also undergo significant

conformational changes upon activation, reducing the size of the entrance to the pocket1.

Figure 1: Catalytic domain of human plasminogen. Figure produced by Chimera2


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Substrate-enzyme interactions: The authors have also described the possibility of plasminogen as

a substrate for tissue-type plasminogen activator (tPa) despite the lack of a crystal structure for

the complex. A ternary inhibitor Pm-SAK-Pm complex was used as a model to study Plg as a

substrate for various plasminogen activators. SAK is a staphylokinase which complexes with Pm

to function as an activator, while the second Pm approximates the activation product.

Substituting the activator Pm in the ternary complex with tPa and activation product Pm with

Plg, the authors saw that some displacement was required in order to bring the activation loop to

the appropriate position for hydrolyzation by tPa. The activation loop of Plg consists of

approximately 10 residues and, as previously stated, the scissile bond between Arg561 and Val562

is cleaved upon activation. The following nomenclature will be used to describe the interactions

between the activation loop of Plg and tPa:

P3 -P2 - P1 /P'1- P'2- P'3

Pro-Gly-Arg/Val-Val-Gly

559-560-561/562-563-564

Where the /' refers to the scissile bond3.

Modeling studies showed that after the displacement, the side-chain of P1Arg561 was located in

the S1 specificity pocket, forming a salt bridge with Asp189 of tPa. The P1 Arg561 carbonyl group

interacts with the oxyanion hole of tPa. The P2 Gly560 residue was found in the S3/S4 pocket, and

its backbone amide group interacts via a hydrogen bond with the oxygen atom in the carbonyl

group of the Gly216 residue of tPa. van der Waals interactions exist between P3 Pro559 and the

side chains of Tyr99 and Trp215 of tPa. Other interactions exist between P'1 Val562 and His57 of

tPa, and also between P'2 Val563 and the side chains of Tyr151 and Gln192 of tPa. A hydrogen

bond is seen between the amide group of P'3 Gly564 and the hydroxyl group of tPa Tyr151. The

authors surmised that the tPa Gln192 side-chain destroys the network of hydrogen bonds between

the activation loop and the C domain of Pm. The added flexibility allows loop to insert into the

substrate binding site of tPa. The substrate-enzyme complex falls apart when the P'1-P'2 segment

shifts upon proteolytic cleavage. The dissociation can be attributed to the reduction in interaction

between the enzyme and the substrate when this segment shifts1,4.

Comparison with other structures: In an effort to understand the structure and function of

plasminogen fully, Wakeham et al.5 studied plasminogen in complex with streptokinase (SAK), a

potent activator that converts plasminogen to plasmin (PDB ID: 1L4D). 12 deletions were made in

an N-terminal domain loop unique to SAK, spanning residues 45-70. It was discovered that thisloop is responsible for substrate recognition, although it does not participate in active site

formation. The authors found that the deletion mutant interacted with plasminogen in a different

way as compared to previously elucidated structures. Distinct conformational changes in the S1

entrance frame, the oxyanion stabilizing loop and the autolysis loop of plasminogen were seen.

Disruption of the hydrogen bond between Asp740 and Gly686 was seen as a result of the change

in the autolysis loop.


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Lamba et al3. studied a complex of plasminogen activator with benzamidine (PDB ID: 1RTF), an

active site inhibitor of tPa. The molecule sits tightly within the active site of the enzyme, forming a

two O-two N salt bridge with a Gly and Asp residue, as well as to a solvent water molecule. This

crystal structure showed that benzamidine is an effective inhibitor of tPa. Future systems to study

could include a ternary benzamidine-tPa-plasminogen complex. Knowledge of this structure could

help in designing drugs to cure clotting diseases.

Western blotFrom Wikipedia, the free encyclopedia

Western blot using an antibody that recognizes proteins modified withlipoic acid.
http://en.wikipedia.org/wiki/Lipoic_acidhttp://en.wikipedia.org/wiki/Lipoic_acidhttp://en.wikipedia.org/wiki/Lipoic_acidhttp://en.wikipedia.org/wiki/File:Anti-lipoic_acid_immunoblot.pnghttp://en.wikipedia.org/wiki/File:Anti-lipoic_acid_immunoblot.pnghttp://en.wikipedia.org/wiki/File:Anti-lipoic_acid_immunoblot.pnghttp://en.wikipedia.org/wiki/File:Anti-lipoic_acid_immunoblot.pnghttp://en.wikipedia.org/wiki/File:Anti-lipoic_acid_immunoblot.pnghttp://en.wikipedia.org/wiki/File:Anti-lipoic_acid_immunoblot.pnghttp://en.wikipedia.org/wiki/Lipoic_acid


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The western blot(sometimes called the protein immunoblot) is a widely usedanalyticaltechniqueused to detect specificproteinsin a sample of tissue homogenate or extract. Itusesgel electrophoresisto separate native proteins by 3-D structure or denatured proteins bythe length of the polypeptide. The proteins are then transferred to a membrane(typicallynitrocelluloseorPVDF), where they are stained withantibodiesspecific to the targetprotein.

[1][2]The gel electrophoresis step is included in Western blot analysis to resolve the issue

of the cross-reactivity of antibodies. An improved immunoblot method,Zestern analysis,is ableto address this issue without the electrophoresis step, thus significantly improving the efficiencyof protein analysis.

There are now many reagent companies that specialize in providing antibodies(bothmonoclonalandpolyclonalantibodies) against tens of thousands of differentproteins.

[3]Commercial antibodies can be expensive, although the unbound antibody can be

reused between experiments. This method is used in the fields ofmolecularbiology,biochemistry,immunogeneticsand other molecular biology disciplines.

Other related techniques include dot blot analysis,zestern analysis,immunohistochemistrywhere antibodies are used to detect proteins in tissues and cells byimmunostainingandenzyme-linked immunosorbent assay (ELISA).

The method originated in the laboratory of Harry Towbin at theFriedrich Miescher Institute.[1]Thename western blotwas given to the technique by W. Neal Burnette

[4]and is a play on the

nameSouthern blot,a technique forDNAdetection developed earlier byEdwin Southern.Detection of RNA is termednorthern blotand was developed by George Stark atStanford.

[5]

Contents

[hide]

1 Steps

o 1.1 Tissue preparation

o 1.2 Gel electrophoresis

o 1.3 Transfer

o 1.4 Blocking

o 1.5 Detection

1.5.1 Two steps

1.5.2 One step

o 1.6 Analysis

1.6.1 Colorimetric detection

1.6.2 Chemiluminescent detection

1.6.3 Radioactive detection

1.6.4 Fluorescent detection

o 1.7 Secondary probing

2 2-D gel electrophoresis

3 Medical diagnostic applications

4 Protocols

5 See also

6 References
http://en.wikipedia.org/wiki/Analytical_techniquehttp://en.wikipedia.org/wiki/Analytical_techniquehttp://en.wikipedia.org/wiki/Analytical_techniquehttp://en.wikipedia.org/wiki/Analytical_techniquehttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/PVDFhttp://en.wikipedia.org/wiki/PVDFhttp://en.wikipedia.org/wiki/PVDFhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Zestern_analysishttp://en.wikipedia.org/wiki/Zestern_analysishttp://en.wikipedia.org/wiki/Zestern_analysishttp://en.wikipedia.org/wiki/Monoclonal_antibodieshttp://en.wikipedia.org/wiki/Monoclonal_antibodieshttp://en.wikipedia.org/wiki/Monoclonal_antibodieshttp://en.wikipedia.org/wiki/Polyclonal_antibodieshttp://en.wikipedia.org/wiki/Polyclonal_antibodieshttp://en.wikipedia.org/wiki/Polyclonal_antibodieshttp://en.wikipedia.org/wiki/Western_blot#cite_note-urlExactantigen-3http://en.wikipedia.org/wiki/Western_blot#cite_note-urlExactantigen-3http://en.wikipedia.org/wiki/Western_blot#cite_note-urlExactantigen-3http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Immunogeneticshttp://en.wikipedia.org/wiki/Immunogeneticshttp://en.wikipedia.org/wiki/Immunogeneticshttp://en.wikipedia.org/wiki/Zestern_analysishttp://en.wikipedia.org/wiki/Zestern_analysishttp://en.wikipedia.org/wiki/Zestern_analysishttp://en.wikipedia.org/wiki/Immunostaininghttp://en.wikipedia.org/wiki/Immunostaininghttp://en.wikipedia.org/wiki/Immunostaininghttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/Friedrich_Miescher_Institute_for_Biomedical_Researchhttp://en.wikipedia.org/wiki/Friedrich_Miescher_Institute_for_Biomedical_Researchhttp://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Western_blot#cite_note-Burnette1981-4http://en.wikipedia.org/wiki/Western_blot#cite_note-Burnette1981-4http://en.wikipedia.org/wiki/Southern_blothttp://en.wikipedia.org/wiki/Southern_blothttp://en.wikipedia.org/wiki/Southern_blothttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Edwin_Southernhttp://en.wikipedia.org/wiki/Edwin_Southernhttp://en.wikipedia.org/wiki/Edwin_Southernhttp://en.wikipedia.org/wiki/Northern_blothttp://en.wikipedia.org/wiki/Northern_blothttp://en.wikipedia.org/wiki/Northern_blothttp://en.wikipedia.org/wiki/Stanford_Universityhttp://en.wikipedia.org/wiki/Stanford_Universityhttp://en.wikipedia.org/wiki/Western_blot#cite_note-Stark1977-5http://en.wikipedia.org/wiki/Western_blot#cite_note-Stark1977-5http://en.wikipedia.org/wiki/Western_blot#cite_note-Stark1977-5http://en.wikipedia.org/wiki/Western_blothttp://en.wikipedia.org/wiki/Western_blothttp://en.wikipedia.org/wiki/Western_blothttp://en.wikipedia.org/wiki/Western_blot#Stepshttp://en.wikipedia.org/wiki/Western_blot#Stepshttp://en.wikipedia.org/wiki/Western_blot#Tissue_preparationhttp://en.wikipedia.org/wiki/Western_blot#Tissue_preparationhttp://en.wikipedia.org/wiki/Western_blot#Gel_electrophoresishttp://en.wikipedia.org/wiki/Western_blot#Gel_electrophoresishttp://en.wikipedia.org/wiki/Western_blot#Transferhttp://en.wikipedia.org/wiki/Western_blot#Transferhttp://en.wikipedia.org/wiki/Western_blot#Blockinghttp://en.wikipedia.org/wiki/Western_blot#Blockinghttp://en.wikipedia.org/wiki/Western_blot#Detectionhttp://en.wikipedia.org/wiki/Western_blot#Detectionhttp://en.wikipedia.org/wiki/Western_blot#Two_stepshttp://en.wikipedia.org/wiki/Western_blot#Two_stepshttp://en.wikipedia.org/wiki/Western_blot#One_stephttp://en.wikipedia.org/wiki/Western_blot#One_stephttp://en.wikipedia.org/wiki/Western_blot#Analysishttp://en.wikipedia.org/wiki/Western_blot#Analysishttp://en.wikipedia.org/wiki/Western_blot#Colorimetric_detectionhttp://en.wikipedia.org/wiki/Western_blot#Colorimetric_detectionhttp://en.wikipedia.org/wiki/Western_blot#Chemiluminescent_detectionhttp://en.wikipedia.org/wiki/Western_blot#Chemiluminescent_detectionhttp://en.wikipedia.org/wiki/Western_blot#Radioactive_detectionhttp://en.wikipedia.org/wiki/Western_blot#Radioactive_detectionhttp://en.wikipedia.org/wiki/Western_blot#Fluorescent_detectionhttp://en.wikipedia.org/wiki/Western_blot#Fluorescent_detectionhttp://en.wikipedia.org/wiki/Western_blot#Secondary_probinghttp://en.wikipedia.org/wiki/Western_blot#Secondary_probinghttp://en.wikipedia.org/wiki/Western_blot#2-D_gel_electrophoresishttp://en.wikipedia.org/wiki/Western_blot#2-D_gel_electrophoresishttp://en.wikipedia.org/wiki/Western_blot#Medical_diagnostic_applicationshttp://en.wikipedia.org/wiki/Western_blot#Medical_diagnostic_applicationshttp://en.wikipedia.org/wiki/Western_blot#Protocolshttp://en.wikipedia.org/wiki/Western_blot#Protocolshttp://en.wikipedia.org/wiki/Western_blot#See_alsohttp://en.wikipedia.org/wiki/Western_blot#See_alsohttp://en.wikipedia.org/wiki/Western_blot#Referenceshttp://en.wikipedia.org/wiki/Western_blot#Referenceshttp://en.wikipedia.org/wiki/Western_blot#Referenceshttp://en.wikipedia.org/wiki/Western_blot#See_alsohttp://en.wikipedia.org/wiki/Western_blot#Protocolshttp://en.wikipedia.org/wiki/Western_blot#Medical_diagnostic_applicationshttp://en.wikipedia.org/wiki/Western_blot#2-D_gel_electrophoresishttp://en.wikipedia.org/wiki/Western_blot#Secondary_probinghttp://en.wikipedia.org/wiki/Western_blot#Fluorescent_detectionhttp://en.wikipedia.org/wiki/Western_blot#Radioactive_detectionhttp://en.wikipedia.org/wiki/Western_blot#Chemiluminescent_detectionhttp://en.wikipedia.org/wiki/Western_blot#Colorimetric_detectionhttp://en.wikipedia.org/wiki/Western_blot#Analysishttp://en.wikipedia.org/wiki/Western_blot#One_stephttp://en.wikipedia.org/wiki/Western_blot#Two_stepshttp://en.wikipedia.org/wiki/Western_blot#Detectionhttp://en.wikipedia.org/wiki/Western_blot#Blockinghttp://en.wikipedia.org/wiki/Western_blot#Transferhttp://en.wikipedia.org/wiki/Western_blot#Gel_electrophoresishttp://en.wikipedia.org/wiki/Western_blot#Tissue_preparationhttp://en.wikipedia.org/wiki/Western_blot#Stepshttp://en.wikipedia.org/wiki/Western_blothttp://en.wikipedia.org/wiki/Western_blot#cite_note-Stark1977-5http://en.wikipedia.org/wiki/Stanford_Universityhttp://en.wikipedia.org/wiki/Northern_blothttp://en.wikipedia.org/wiki/Edwin_Southernhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Southern_blothttp://en.wikipedia.org/wiki/Western_blot#cite_note-Burnette1981-4http://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Friedrich_Miescher_Institute_for_Biomedical_Researchhttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/Immunostaininghttp://en.wikipedia.org/wiki/Zestern_analysishttp://en.wikipedia.org/wiki/Immunogeneticshttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Western_blot#cite_note-urlExactantigen-3http://en.wikipedia.org/wiki/Polyclonal_antibodieshttp://en.wikipedia.org/wiki/Monoclonal_antibodieshttp://en.wikipedia.org/wiki/Zestern_analysishttp://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Western_blot#cite_note-Towbin1979-1http://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/PVDFhttp://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Analytical_techniquehttp://en.wikipedia.org/wiki/Analytical_technique


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7 External links

Steps[edit]Tissue preparation[edit]

Samples can be taken from whole tissue or from cell culture. Solid tissues are first broken downmechanically using ablender(for larger sample volumes), using ahomogenizer(smallervolumes), or bysonication.Cells may also be broken open by one of the above mechanicalmethods. However, virus or environmental samples can be the source of protein and thuswestern blotting is not restricted to cellular studies only.

Assorteddetergents,salts, andbuffersmay be employed to encouragelysisof cells and tosolubilize proteins.Proteaseandphosphataseinhibitors are often added to prevent the digestionof the sample by its own enzymes. Tissue preparation is often done at cold temperatures toavoidprotein denaturingand degradation.

A combination of biochemical and mechanical techniquescomprising various types of filtrationandcentrifugationcan be used to separate different cell compartments andorganelles.

Gel electrophoresis[edit]

Main article:Gel electrophoresis

The proteins of the sample are separated usinggel electrophoresis.Separation of proteins maybe byisoelectric point(pI),molecular weight,electric charge, or a combination of these factors.The nature of the separation depends on the treatment of the sample and the nature of the gel.This is a very useful way to identify a protein.

By far the most common type of gel electrophoresis employspolyacrylamidegels and buffersloaded withsodium dodecyl sulfate(SDS).SDS-PAGE(SDS polyacrylamide gel electrophoresis)maintains polypeptides in a denatured state once they have been treated with strong reducingagents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups[SH and SH]) and thus allows separation of proteins by their molecular weight. Sampled proteinsbecome covered in the negatively charged SDS and move to the positively charged electrodethrough theacrylamidemesh of the gel. Smaller proteins migrate faster through this mesh andthe proteins are thus separated according to size (usually measured in kilodaltons,kDa). Theconcentration of acrylamide determines the resolution of the gel - the greater the acrylamideconcentration the better the resolution of lower molecular weight proteins. The lower theacrylamide concentration the better the resolution of higher molecular weight proteins. Proteinstravel only in one dimension along the gel for most blots.

Samples are loaded into wellsin the gel. One lane is usually reserved for a markeror ladder, acommercially available mixture of proteins having defined molecular weights, typically stained so
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as to form visible, coloured bands. Whenvoltageis applied along the gel, proteins migratethrough it at different speeds dependent on their size. These different rates of advancement(differentelectrophoretic mobilities)separate into bandswithin each lane.

It is also possible to use atwo-dimensional (2-D) gelwhich spreads the proteins from a singlesample out in two dimensions. Proteins are separated according to isoelectric point (pH at whichthey have neutral net charge) in the first dimension, and according to their molecular weight inthe second dimension.

Transfer[edit]

In order to make the proteins accessible to antibody detection they are moved from within the gelonto a membrane made ofnitrocelluloseorpolyvinylidene difluoride(PVDF). The primary

method for transferring the proteins is calledelectroblottingand uses an electric current to pullproteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from withinthe gel onto the membrane while maintaining the organization they had within the gel. An oldermethod of transfer involves placing a membrane on top of the gel, and a stack of filter papers ontop of that. The entire stack is placed in a buffer solution which moves up the paper bycapillaryaction,bringing the proteins with it. In practice this method is not used as it takes too much time;electroblotting is preferred. As a result of either "blotting" process, the proteins are exposed on athin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is basedupon hydrophobic interactions, as well as charged interactions between the membrane andprotein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do notstand up well to repeated probings.
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The uniformity and overall effectiveness of transfer of protein from the gel to the membrane canbe checked by staining the membrane withCoomassie Brilliant BlueorPonceau Sdyes.Ponceau S is the more common of the two, due to its higher sensitivity and water solubility, thelatter making it easier to subsequently destain and probe the membrane, as described below.

[6]

Blocking[edit]

Since the membrane has been chosen for its ability to bind protein and as both antibodies andthe target are proteins, steps must be taken to prevent the interactions between the membraneand the antibody used for detection of the target protein. Blocking of non-specific binding isachieved by placing the membrane in a dilute solution of protein - typically 3-5%Bovine serumalbumin(BSA) ornon-fat dry milk(both are inexpensive) inTris-Buffered Saline(TBS) or I-Block,

with a minute percentage (0.1%) of detergent such asTween 20orTriton X-100.The protein inthe dilute solution attaches to the membrane in all places where the target proteins have notattached. Thus, when the antibody is added, there is no room on the membrane for it to attachother than on the binding sites of the specific target protein. This reduces "noise" in the finalproduct of the western blot, leading to clearer results, and eliminates false positives.

Detection[edit]

During the detection process the membrane is "probed" for the protein of interest with a modifiedantibody which is linked to a reporter enzyme; when exposed to an appropriate substrate thisenzyme drives a colourimetric reaction and produces a color. For a variety of reasons, thistraditionally takes place in a two-step process, although there are now one-step detectionmethods available for certain applications.

Two steps[edit]

Primary antibody

The primary antibodies are generated when a host species or immune cell culture is exposed toprotein of interest (or a part thereof). Normally, this is part of the immune response, whereashere they are harvested and used as sensitive and specific detection tools that bind the proteindirectly.

After blocking, a dilute solution of primary antibody (generally between 0.5 and 5micrograms/mL) is incubated with the membrane under gentle agitation. Typically, the solution iscomposed of buffered saline solution with a small percentage of detergent, and sometimes with

powdered milk or BSA. The antibody solution and the membrane can be sealed and incubatedtogether for anywhere from 30 minutes to overnight. It can also be incubated at different
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temperatures, with higher temperatures being associated with more binding, both specific (to thetarget protein, the "signal") and non-specific ("noise").

Secondary antibody

After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to

another antibody, directed at a species-specific portion of the primary antibody. Antibodies comefrom animal sources (or animal sourcedhybridomacultures); an anti-mouse secondary will bindto almost any mouse-sourced primary antibody, which allows some cost savings by allowing anentire lab to share a single source of mass-produced antibody, and provides far more consistentresults. This is known as a secondary antibody, and due to its targeting properties, tends to bereferred to as "anti-mouse," "anti-goat," etc. The secondary antibody is usually linked tobiotinorto a reporterenzymesuch asalkaline phosphataseorhorseradish peroxidase.This means thatseveral secondary antibodies will bind to one primary antibody and enhance the signal.

Most commonly, ahorseradish peroxidase-linked secondary is used to cleave achemiluminescent agent, and the reaction product producesluminescencein proportion to theamount of protein. A sensitive sheet of photographic film is placed against the membrane, andexposure to the light from the reaction creates an image of the antibodies bound to the blot. Acheaper but less sensitive approach utilizes a4-chloronaphtholstain with 1%hydrogen peroxide;reaction of peroxide radicals with 4-chloronaphthol produces a dark purple stain that can bephotographed without using specialized photographic film.

As with theELISPOTandELISAprocedures, the enzyme can be provided with a substratemolecule that will be converted by the enzyme to a colored reaction product that will be visible on

the membrane (see the figure below with blue bands).

Another method of secondary antibody detection utilizes a near-infrared (NIR) fluorophore-linkedantibody. Light produced from the excitation of a fluorescent dye is static, making fluorescentdetection a more precise and accurate measure of the difference in signal produced by labeledantibodies bound to proteins on a western blot. Proteins can be accurately quantified becausethe signal generated by the different amounts of proteins on the membranes is measured in astatic state, as compared to chemiluminescence, in which light is measured in a dynamic state.

[7]

A third alternative is to use a radioactive label rather than an enzyme coupled to the secondaryantibody, such as labeling an antibody-binding protein likeStaphylococcusProtein A orStreptavidin with a radioactive isotope of iodine. Since other methods are safer, quicker, andcheaper, this method is now rarely used; however, an advantage of this approach is the

sensitivity of auto-radiography based imaging, which enables highly accurate proteinquantification when combined with optical software (e.g. Optiquant).
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One step[edit]

Historically, the probing process was performed in two steps because of the relative ease ofproducing primary and secondary antibodies in separate processes. This gives researchers andcorporations huge advantages in terms of flexibility, and adds an amplification step to thedetection process. Given the advent of high-throughput protein analysis and lower limits of

detection, however, there has been interest in developing one-step probing systems that wouldallow the process to occur faster and with fewer consumables. This requires a probe antibodywhich both recognizes the protein of interest and contains a detectable label, probes which areoften available for knownprotein tags.The primary probe is incubated with the membrane in amanner similar to that for the primary antibody in a two-step process, and then is ready for directdetection after a series of wash steps.

Western blot using radioactive detection system

Analysis[edit]

After the unbound probes are washed away, the western blot is ready for detection of the probesthat are labeled and bound to the protein of interest. In practical terms, not all westerns revealprotein only at one band in a membrane. Size approximations are taken by comparing thestained bands to that of the marker or ladder loaded during electrophoresis. The process isrepeated for a structural protein, such as actin or tubulin, that should not change betweensamples. The amount of target protein isnormalizedto the structural protein to control betweengroups. This practice ensures correction for the amount of total protein on the membrane in caseof errors or incomplete transfers.

Color imetr ic detect ion[edit]

The colorimetric detection method depends on incubation of the western blot with a substratethat reacts with the reporter enzyme (such asperoxidase)that is bound to the secondaryantibody. This converts the soluble dye into an insoluble form of a different color that precipitatesnext to the enzyme and thereby stains the membrane. Development of the blot is then stoppedby washing away the soluble dye. Protein levels are evaluated throughdensitometry(howintense the stain is) orspectrophotometry.

Chemiluminescent detect ion[edit]

Chemiluminescentdetection methods depend on incubation of the western blot with a substratethat will luminesce when exposed to the reporter on the secondary antibody. The light is thendetected by photographic film, and more recently by CCD cameras which capture a digital imageof the western blot. The image is analysed by densitometry, which evaluates the relative amountof protein staining and quantifies the results in terms of optical density. Newer software allowsfurther data analysis such as molecular weight analysis if appropriate standards are used.
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Radioactiv e detection[edit]

Radioactive labels do not require enzyme substrates, but rather allow the placement of medicalX-ray film directly against the western blot which develops as it is exposed to the label andcreates dark regions which correspond to the protein bands of interest (see image to the right).The importance of radioactive detections methods is declining due to its hazardousradiation

[citation needed], because it is very expensive, health and safety risks are high, and ECL

(enhanced chemiluminescence) provides a useful alternative.

Fluorescent detect ion[edit]

The fluorescently labeled probe is excited by light and the emission of the excitation is thendetected by a photosensor such as CCD camera equipped with appropriate emission filterswhich captures a digital image of the western blot and allows further data analysis such asmolecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to

be one of the best methods for quantification, but is less sensitive than chemiluminescence.[8]

Secondary probing[edit]

One major difference between nitrocellulose and PVDF membranes relates to the ability of eachto support "stripping" antibodies off and reusing the membrane for subsequent antibody probes.While there are well-established protocols available for stripping nitrocellulose membranes, thesturdier PVDF allows for easier stripping, and for more reuse before background noise limitsexperiments. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95%ethanol, isopropanol or methanol before use. PVDF membranes also tend to be thicker and moreresistant to damage during use.

2-D gel electrophoresis[edit]Main article:Two-dimensional gel electrophoresis

2-dimensional SDS-PAGE uses the principles and techniques outlined above. 2-D SDS-PAGE,as the name suggests, involves the migration of polypeptides in 2 dimensions. For example, inthe first dimension polypeptides are separated according toisoelectric point,while in the seconddimension polypeptides are separated according to theirmolecular weight.The isoelectric pointof a given protein is determined by the relative number of positively (e.g. lysine and arginine) andnegatively (e.g. glutamate and aspartate) charged amino acids, with negatively charged aminoacids contributing to a low isoelectric point and positively charged amino acids contributing to ahigh isoelectric point. Samples could also be separated first under nonreducing conditions usingSDS-PAGE and under reducing conditions in the second dimension, which breaks apart disulfide

bonds that hold subunits together. SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel.
http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=12http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=12http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=12http://en.wikipedia.org/wiki/Wikipedia:Citation_neededhttp://en.wikipedia.org/wiki/Wikipedia:Citation_neededhttp://en.wikipedia.org/wiki/Wikipedia:Citation_neededhttp://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=13http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=13http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=13http://en.wikipedia.org/wiki/Western_blot#cite_note-8http://en.wikipedia.org/wiki/Western_blot#cite_note-8http://en.wikipedia.org/wiki/Western_blot#cite_note-8http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=14http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=14http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=14http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=15http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=15http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=15http://en.wikipedia.org/wiki/Two-dimensional_gel_electrophoresishttp://en.wikipedia.org/wiki/Two-dimensional_gel_electrophoresishttp://en.wikipedia.org/wiki/Two-dimensional_gel_electrophoresishttp://en.wikipedia.org/wiki/Isoelectric_pointhttp://en.wikipedia.org/wiki/Isoelectric_pointhttp://en.wikipedia.org/wiki/Isoelectric_pointhttp://en.wikipedia.org/wiki/Molecular_weighthttp://en.wikipedia.org/wiki/Molecular_weighthttp://en.wikipedia.org/wiki/Molecular_weighthttp://en.wikipedia.org/wiki/File:Western_blot_chemiluminescent_detection.pnghttp://en.wikipedia.org/wiki/Molecular_weighthttp://en.wikipedia.org/wiki/Isoelectric_pointhttp://en.wikipedia.org/wiki/Two-dimensional_gel_electrophoresishttp://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=15http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=14http://en.wikipedia.org/wiki/Western_blot#cite_note-8http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=13http://en.wikipedia.org/wiki/Wikipedia:Citation_neededhttp://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=12


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In principle, this method allows for the separation of all cellular proteins on a single large gel. Amajor advantage of this method is that it often distinguishes between differentisoformsof aparticular protein - e.g. a protein that has been phosphorylated (by addition of a negativelycharged group). Proteins that have been separated can be cut out of the gel and then analysedbymass spectrometry,which identifies the protein.

Please refer to reference articles for examples of the application of 2-D SDS PAGE.

Medical diagnostic applications[edit]

The confirmatoryHIV testemploys a western blot to detect anti-HIV antibody in a

humanserumsample. Proteins from knownHIV-infected cells are separated and blotted on

a membrane as above. Then, the serum to be tested is applied in the primary antibody

incubation step; free antibody is washed away, and a secondary anti-human antibody linked

to an enzyme signal is added. The stained bands then indicate the proteins to which the

patient's serum contains antibody.

A western blot is also used as the definitive test forBovine spongiform

encephalopathy(BSE, commonly referred to as 'mad cow disease').

Some forms ofLyme diseasetesting employ western blotting.

Western blot can also be used as a confirmatory test for Hepatitis B infection.

In veterinary medicine, western blot is sometimes used to confirmFIV+ status in cats.
http://en.wikipedia.org/wiki/Isoformshttp://en.wikipedia.org/wiki/Isoformshttp://en.wikipedia.org/wiki/Isoformshttp://en.wikipedia.org/wiki/Mass_spectrometryhttp://en.wikipedia.org/wiki/Mass_spectrometryhttp://en.wikipedia.org/wiki/Mass_spectrometryhttp://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=16http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=16http://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=16http://en.wikipedia.org/wiki/HIV_testhttp://en.wikipedia.org/wiki/HIV_testhttp://en.wikipedia.org/wiki/HIV_testhttp://en.wikipedia.org/wiki/Blood_plasmahttp://en.wikipedia.org/wiki/Blood_plasmahttp://en.wikipedia.org/wiki/Blood_plasmahttp://en.wikipedia.org/wiki/HIVhttp://en.wikipedia.org/wiki/HIVhttp://en.wikipedia.org/wiki/HIVhttp://en.wikipedia.org/wiki/Bovine_spongiform_encephalopathyhttp://en.wikipedia.org/wiki/Bovine_spongiform_encephalopathyhttp://en.wikipedia.org/wiki/Bovine_spongiform_encephalopathyhttp://en.wikipedia.org/wiki/Bovine_spongiform_encephalopathyhttp://en.wikipedia.org/wiki/Lyme_diseasehttp://en.wikipedia.org/wiki/Lyme_diseasehttp://en.wikipedia.org/wiki/Lyme_diseasehttp://en.wikipedia.org/wiki/FIVhttp://en.wikipedia.org/wiki/FIVhttp://en.wikipedia.org/wiki/FIVhttp://en.wikipedia.org/wiki/Lyme_diseasehttp://en.wikipedia.org/wiki/Bovine_spongiform_encephalopathyhttp://en.wikipedia.org/wiki/Bovine_spongiform_encephalopathyhttp://en.wikipedia.org/wiki/HIVhttp://en.wikipedia.org/wiki/Blood_plasmahttp://en.wikipedia.org/wiki/HIV_testhttp://en.wikipedia.org/w/index.php?title=Western_blot&action=edit&section=16http://en.wikipedia.org/wiki/Mass_spectrometryhttp://en.wikipedia.org/wiki/Isoforms

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