Green Fluorescent Protein Chromatography Lab Report

The proteins were visualized using the Coomassie Blue stain. Coomassie Blue binds non-specifically and nearly stoichiometrically to all proteins [5]. Proteins have a higher affinity to the dye than the polyacrylamide gel; therefore, after removing excess stain, the protein bands can be visualized [4]. This non-selective binding was essential for analyzing the purity in each invertase fraction, as both non-target proteins and invertase were visualized. As shown in Fig. 1., excluding invertase fraction 2, the general trend was that the number of protein bands decreased from fractions 1 to 4, while a band with a molecular weight around 1.2 x 102 kDa became more prominent and intense. These results were expected for a successful purification [4]. During experiment 6, it was observed that the specific activity increased during each successive fraction. Since the same amount of protein, from each invertase

Green fluorescent protein (GFP) comes from the jellyfish Aequorea Victoria is rare proteins with high fluoresce and absorbance. The purpose this experiments is to purify and express a His2-tagged recombinant from of GFP (rGFP) from the E. coli strain BL21(DE3)< pRSETA-GFPUV > through a series of experiments by using Ni+2 agarose affinity chromatography technology. The GFPuv gene (UV-optimized GFP) was over expressed in the E. Coil strain BL21 (DE3) (pLysS) as an n-terminal His6/Xpress epitope tagged bind protein. Then using Ni2+ Agarose affinity chromatography to obtain purification of the crude extract. Then observe under the long wavelength UV light, the activity of the rGFP in the column fraction. Bradford assay was performed to obtain the total protein amount. When calculating the

Green Fluorescent Protein, produced by the bioluminescent jellyfish Aequorea victoria, is a protein that fluoresces green under ultraviolet light. Since its discovery, properties of the protein have been improved by mutations in the gene resulting in the expansion of its spectrum, which now contains brighter variants and multiple different colors. GFP is used in a wide variety of applications and technologies. Its many different applications have contributed greatly, and continue to do so, in numerous fields of study including, but not limited to, cellular and molecular biology, microbiology, biotechnology, and medicine.

Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).

Green Fluorescent Protein (GFP) is a fluorescent protein found in jellyfish that causes organisms to fluoresce. This protein was the first fluorescent protein to be discovered and has been highly useful in a broad range of cell biology disciplines; because of it’s highly useful reporter genes and the ability to use multicolor protein tracking in living cells it has been useful in many scientific experiments such as E. Coli transformations. There have been many other fluorescent proteins that have been cloned from a wide variety of marine invertebrates, therefore making GFP not the only standing fluorescent protein present. (Shaner 2014). The scientific uses of GFP have included

Traditional reagent strip testing for protein uses the principle of the protein error of indicators to produce a visible colorimetric reaction. The protein (primarily albumin) accepts hydrogen ions from the indicator causing change in color. (Susan King Strasinger, 2008)

The five reagents that were used in this lab were Ninhydrin, Biuret, Orcinol, I2KI, and Nile blue. Ninhydrin in solution will react with amino acids producing a blue product. In the absence of amino acids, nope color develops. this test is both qualitative and quantitative. the development of a blue color indicates the presence or absence of amino acids and the intensity of that color reflects the concentration of amino acids present in the sample being tested. the next reagent that was used was the biuret reagent. this region is used to test for the presence of proteins. when an aqueous sample (using water as the solvent) is mixed with this reagent, the appearance of a purple color indicates that protein is present in the sample. in the absence of proteins, the simple commands light

The experiments that were completed previously offered a comprehensive understanding of how rGFP was induced, expressed, and purified. To outline, Ni2+-agarose affinity chromatography was done to separate the protein of interest through a strong affinity to the His-6 tag in the rGFP to the column. The Bradford assay is where the estimation of the amount protein of the samples was done. Then the SDS-PAGe gel showed an estimation of the molecular weight and purity of samples. This was important in identifying the protein. Finally developed a Western Blot, confirming the presence of rGFP through band

In order to create this revolutionary new fluorescent visualization protein, the researchers had to test it in many

They are key constituents of all biological systems, and perform a great variety of functional and structural roles. Proteins play a crucial role in almost all biological processes, like signal transmission, catalysis, and structural support. This important range of functions comes from the existence of thousands of proteins, which are each folded into a characteristics three-dimensional structure, the same structure that allows it to interact with one or perhaps more molecules. A lot of the functional and structural studies of proteins are conducted with purified preparations of proteins. In general the purification methods of proteins aim to exploit the differences in their different properties such as, solubility, size, charge, and resin-binding specificity, all with the purpose to enrich the solution for the targeted protein

The plasmid first had to lose S65T-GFP so it could take up yEGFP. This was done by adding Pac1 and Asc1 to the plasmid to cut out the gene and leave the proper sticky ends for yEGFP to ligate to. A gel electrophoresis was then used to separate pWDH444 and S65T-GFP. The pWDH444 was cut from the gel electrophoresis and removed from the gel. After successful removal of the plasmid from the gel pWDH444 and yEGFP were ligated to create a new plasmid with the ideal GFP. The new plasmid, pWDH444-yEGFP was the ideal plasmid for yeast due to the enhanced GFP with a codon bias for yeast and it had double the light output of S65T-GFP. The difference in fluorescence was detected using a extra sensitive fluorimeter than normal and 495-nm glass, sharp cut-off filter. The filter reduced the amount of light shattering leading to an improved sensitive reading from the

During the digestion of proteins experiment four test tubes were used each one containing albumin while only two contained hydrochloric acid. Table 1. Digestion of Proteins Tube Color 1 Light Purple with blue 2 Dark purple 3 No change 4

In many cases, the proteins in question are monoclonal antibodies generated against a particular target of interest such as a cell surface receptor, a cytoskeletal protein, or a cellular organelle. In some cases, a fluorescent label is used to track the fate of a particular protein as it is processed in the ER and Golgi complex, transported along microtubules, or secreted from the cell altogether. In still other cases, two or more fluorescently tagged proteins are introduced into the same cell to see whether they localize in the same or separate compartments or organelles.

SDS-PAGE separates proteins according to their size. Sodium dodecyl sulfate (SDS) dissolves hydrophobic molecules and carries a negative charge. A cell incubated with SDS would have its membranes dissolve and all the proteins becoming soluble and covered with negative charges. As a result, all the proteins only have their primary structures and a large negative charge allowing them to migrate towards the positive pole of an electric field. Polyacylamide gel is used to separate proteins according to their sizes. The gel has pores of different sizes that act as obstacles for the proteins to pass through. Switch off the current, stain the proteins, and the end result would be bands of protein separated according to their molecular weight. One

GFP was first isolated in in the 1970’s from the jellyfish Aequorea victoria. Scientist were able to use the genetic engineering to introduce fluorescent proteins into living organisms once identifying its specific DNA sequence. Osamu Shimomura, Martin Chalfie and Roger Tsien discovered the different changes in the patterns of absorption and emission in order to develop a rainbow fluorescent protein. At this time, the scientists notices that amino acid substitutions in GFP changed the behavior of the protein responsible for light production or a chromophore. The GFP and its related fluorescent proteins have become an essential tool in cell and molecular biology. proteins can be tagged with fluorescent proteins and then expressed in cells using DNA cloning strategies. Scientist regulate the expression of recombinant proteins using an inducible promoter or a genetic on / off switch. these sequences allow precise control because expression of the gym only turn on in the presence of a small molecule. In this experiment, and inducible promoter is used to regulate GFP