FISH-SDSall - V.Woriax 9/01 BIO 3710 - Cell Biology 12%...

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V.Woriax 9/01 BIO 3710 -- Cell Biology – 12% SDS-PAGE SETUP FOR FISH PROTEINS FINGERPRINTING -- DAY I-III Purpose : Prepare a denatured polyacrylamide gel system; separate a mixture of proteins according to molecular weight; devise a standard curve; determine the molecular weight of unknown proteins. Precautions : Acrylamide and bis-acrylamide are neurotoxins and must be handled carefully. Wear gloves, labcoat, and protective eyewear at all times during the lab. Keep working chemicals on benchkote at all times. TEMED is highly flammable and volatile. Keep capped and take care in dispensing (using hood/snorkels as necessary). Reference: Laemmli, UK (1970) Nature , 227 : 680-685 Technique: SDS-PAGE ( s odium d odecyl s ulfate - p oly a crylamide g el e lectrophoresis) is a technique for characterizing macromolecules and for assaying their purity. The procedure is based on the principle that molecules such as proteins possess a charge and as a result are capable of moving when placed in an electric field. The velocity at which a molecule moves is proportional to the strength of the electric field and the charge on the molecule, but is inversely proportional to the size of the molecule. Polyacrylamide gels are commonly used for electrophoresis of macromolecules. The gels are prepared by polymerization of the acrylamide monomer and the cross-linking co-monomer bis(acrylamide). The polymerization results in cross-linked acrylamide chains. The average pore size is determined by the amount of acrylamide used and the degree of cross-linkage, both of which are parameters that can be varied for optimum separation. SDS gels are denaturing gels. SDS is an anionic (negatively charged) detergent that unfolds (denatures) the native protein and separates subunits. SDS binds uniformly to the hydrophobic regions of the polypeptide, giving a negative charge that is proportional to the length of the polypeptide chain. β -Mercaptoethanol is used to break subunits held together by disulfide bonds, thereby reducing the disulfide linkages to sulfhydryl groups. Since each protein is now uniformly coated with negative charges, different proteins can be separated on the basis of size. DAY I:
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FISH-SDSall - V.Woriax 9/01 BIO 3710 - Cell Biology 12%...

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