Saturday, November 30, 2013

Analysis of Purified rGFP Using SDS-PAGE gel/Coomassie - Blue and Western Blot

ABSTRACTGreen fluorescent protein (GFP) has great potential for biological studies due to its stability, flexibility, and false characteristics. The GFP gene can be cloned and reconfigured to be expressed in numerous organisms such as E. coli to induce its expression for refining and analysis. The rGFP produced can then be purified using Ni+²-agarose rachis and quantified using Bradford assay. Purity analysis of the rGFP obtained was done using SDS-PAGE gelatine/Coomassie Blue. Finally a Western Blot was perform to confirm the protein localize is actually the protein of interest, rGFP. According to these different methods, yield, purity and rGFP activity were conjure up and they were 4.20 ug, 15% and 10200 RFUs, repetitively. INTRODUCTIONGreen fluorescent protein, also known as GFP, is a protein produced by a jellyfish Aequorea Victoria which fluoresces in the kelvin portion of the visible spectrum. GFP is an extremely stable 27kDa protein composed of 238 amino g roup acids, which form a ß-barrel wrapped around a primaeval core. The intrinsic fluorescence of the protein is due to a covalently attached chromophore which is formed by posttranslational intramolecular reactions involving cyclization and oxidation of amino acids Ser ? Tyr ? Gly [1]. By have and incorporating the nine-fold mutations (such as one class of GFP fold and cyclize properly and other(a) class do not )will lead to an increased in fluorescence.
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Poly histidinetag (6xHis tag) is an amino acid motif in proteins that consists of at to the worst degree six Histidine (His) residues, often at the N- or C-terminus of the protein [3]. Histidine tag! ging allows relationship isolation of the protein with attendant proteins without affecting the function of the protein. In swan to sick the rGFP protein, the gene fusion technique was used in which the GFP protein was amalgamate with another protein (poly histidinetag) that is easily purified by Ni+2 ?agarose... If you want to get a full essay, order it on our website: OrderCustomPaper.com

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