Analysis Of Purified R Gfp Using Sds Page Gel/Coomassie Blue And Western Blot

2803 words - 11 pages

ABSTRACTGreen fluorescent protein (GFP) has great potential for biological studies due to its stability, flexibility, and unique characteristics. The GFP gene can be cloned and reconfigured to be expressed in numerous organisms such as E. coli to induce its expression for purification and analysis. The rGFP produced can then be purified using Ni+²-agarose column and quantified using Bradford assay. Purity analysis of the rGFP obtained was done using SDS-PAGE gel/Coomassie Blue. Finally a Western Blot was performed to confirm the protein present is actually the protein of interest, rGFP. According to these different methods, yield, purity and rGFP activity were found 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 green portion of the visible spectrum. GFP is an extremely stable 27kDa protein composed of 238 amino acids, which form a ß-barrel wrapped around a central 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 combining and incorporating the multiple mutations (such as one class of GFP fold and cyclize properly and another class do not )will lead to an increased in fluorescence.Poly histidinetag (6xHis tag) is an amino acid motif in proteins that consists of at least six Histidine (His) residues, often at the N- or C-terminus of the protein [3]. Histidine tagging allows affinity isolation of the protein with attendant proteins without affecting the function of the protein. In order to purify the rGFP protein, the gene fusion technique was used in which the GFP protein was fused with another protein (poly histidinetag) that is easily purified by Ni+2 –agarose affinity chromatography. The tagged protein (rGFP) has affinity for the metal ion nickel, hence it binds to the Ni+²-agarose matrix in the column, and the other impurities that are not bound to the column are washed away. When rGFP is passed through a column containing immobilized nickel, it binds to nickel through His residues. Then the tagged protein is eluted by imidazole which competes and displaces the His-tag from nickel co-ordination, freeing the His-tagged proteins.Through experiments, the expression and purification of rGFP was done successfully. The ease in detection is shown in its ability to fluoresce up to 509nm when excited with ultraviolet light of 395nm [1]. No cofactor is required for fluorescence, which makes it inexpensive for experiments. Real time detection is made possible by shining UV light on the fractions, which will then fluoresce green color to indicate the presence of rGFP. Since GFP can be cloned and configured (recombinant GFP) to be expressed in numerous organisms such as E. coli, only small amounts of...

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