sodium dodecyl sulfate polyacrylamide gel electrophoresis ii

8/9/2019 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis II

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(SDS-PAGE)


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A technique widely used to separateproteins according to their electrophoreticmobility

SDS gel electrophoresis of samples havingidentical charge per unit mass due tobinding of SDS results in fractionation by

size and is probably the world's most widelyused biochemical method


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C 12H 25NaO 4SMW: 288.38

most common dissociating agent used to

denature native proteins toindividual polypeptides

When a protein mixture is heated to 100 C

in presence of SDS, the detergent wrapsaround the polypeptide backbone. It bindsto polypeptides in a constant weight ratio of 1.4 g/g of polypeptide


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SDS is a negatively charged detergent.Disrupts secondary and tertiary protein structuresby breaking hydrogen bonds and unfolding protein.Masks charge on protein so that all proteins actthe same as regards charge.Prevents protein aggregation.Prevents protein shape from influencing gel run.


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Samples may be taken from whole tissue orcell cultureCombination of biochemical and mechanicaltechniques can be used to separate different

cell compartments and organelles The solution of proteins to be analyzed ismixed with SDS, ananionic detergent which denatures secondaryand nondisulfidelinked tertiary structures,and applies a negative charge to each proteinin proportion to its mass


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A tracking dye may be added to the proteinsolution (of a size smaller than protein) toallow the experimenter to track theprogress of the protein solution through thegel during the electrophoretic run


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the gel may be stained allowingvisualization of the separated proteins, orprocessed furtherdifferent proteins will appear as distinctbands within the gel. It is common torun molecular markersof known molecularweight in a separate lane in the gel, in order

to calibrate the gel and determine theweight of unknown proteins by comparingthe distance traveled relative to the marker


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The gel is actually formed because theacrylamide solution contains a small amount.Gel electrophoresis is usually the first choiceas an assay of protein purity due to itsreliability and ease. The presence of SDS andthe denaturing step causes proteins to beseparated solely based on size. Falsenegatives and positives are possible. Acomigrating contaminant can appear as thesame band as the desired protein.


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Sodium Dodecyl Sulfate PolyacrylamideGel Electrophoresis

A procedure to separate proteins anddetermine their Molecular Weights.

It follows the principle that a charged

molecule will migrate in an electric fieldtoward an electrode of opposite sign.


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Electrophoresis in Principle:

Separation of charged molecules in electric field isa function of: Relative mobility of charged species (related tofrictional resistance which is related to size). Charge on the species. Will migrate toward cathode (-) or anode (+). Separation occurs due to different rates of migration due to magnitude of charge andfrictional resistance (related to size).


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Mobility:

Rf = (Z E)f

where Z = charge on molecule E = Voltage applied (driving force) f = frictional resistance

Rf is measured by:Rf = Distance protein band moves

Distance dye front moves


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Extract ProteinSolubilize and Denature ProteinSeparate Proteins on a gel

Stain proteins (visualization)Analyze and interpret results


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Primary structure = linear chain of

amino acids

Secondary structure = domains of repeating structures, such as -pleatedsheets and -helices

Tertiary structure = 3-dimensionalshape of a folded polypeptide,maintained by disulfide bonds,electrostatic interactions, hydrophobiceffects

Quaternary structure = several polypeptide chains associated together to form a functional protein


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SDS (Sodium DodecylSulfate) detergent

solubilizes anddenatures proteins

negative charge toproteins

Heat denatures proteins O S O

O

O

-

CH 2

CH 2

CH 2

CH 2

CH 2

CH 2

CH2

CH 2

CH 2

CH 2

CH 2

CH 3

SDS


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.


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The amino acid cysteine contains asulfhydryl (-SH) group that spontaneously

forms a disulfide bond (-S-S-) with anothersulfhydryl group under normal intracellularconditions. Disulfide bonding is covalentand is not disrupted by SDS.

DTT is a strong reducing agent. Its specificrole in sample denaturation is to removethe last bit of tertiary and quaternarystructure by reducing disulfide bonds.


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Negatively chargedproteins move topositive electrode

Smaller proteinsmove faster

Proteins separateby size -

+

s-s

SDS, heat

proteins withSDS


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Size measured in daltons (Da) or kilodaltons (kDa)Dalton = atomic mass unit

= corresponds to mass of hydrogen

molecule (1.66 x 10 -24 gram)= defined also as 1/16 of the mass of an

atom of oxygen

Average amino acid = 110 Da Average nucleotide pair = 649 Da


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DNADNAProteinsProteins
http://www.microscopy.fsu.edu/primer/anatomy/brightfieldgallery/frogstriatedmusclelarge.html


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Muscles contain many proteins, some of which areMuscles contain many proteins, some of which arevariable from organism to organismvariable from organism to organismActin and MyosinActin and Myosin Form muscle fibers that allow muscles to contract andForm muscle fibers that allow muscles to contract and

relaxrelax Most common muscle proteins in all animalsMost common muscle proteins in all animals


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Lane 1. Kaleidoscope Markers

2. Shark

3. Salmon

4. Trout

5. Catfish

6. Sturgeon

7. Actin and Myosin Standard


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Myosin blue -galactosidase magentaBovine serum albumin greenCarbonic anhydrase violetSoybean trypsin inhibitor orangeLysozyme redAprotinin - blue

Kaleidoscope standardKaleidoscope standard


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Protein kDa Functiontitin 3000 center myosin in sarcomeredystrophin 400 anchoring to plasma

membranefilamin 270 cross-link filaments into gel

myosin heavy chain 210 slide filamentsspectrin 265 attach filaments to plasma

membranenebulin 107 regulate actin assembly -actinin 100 bundle filamentsgelosin 90 fragment filamentsfimbrin 68 bundle filaments

actin 42 form filaments tropomyosin 35 strengthen filaments

myosin light chain 27 slide filamentstroponin (T, I, C) 30, 19, 17 mediate regulation of

contractionthymosin 5 sequester actin

monomers


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# of proteins in common

Total # of unique proteins

100%

Pairwise comparisons between species


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Protein Color MW (daltons) on TrisHCl gel

Myosin Blue 204,649

B-galactosidase Magenta 127,511

Bovine serumalbumin

Green 85,130

Carbonic anhydrase Violet 37,830

Soybean trypsininhibitor

Orange 30,906

Lysozyme Red 27,230

Aprotinin Blue 6,638

sodium dodecyl sulfate polyacrylamide gel electrophoresis ii

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