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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