Lab Report Green Fluorescent Protein

This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.

When faced with a crisis of identity, we often stick with those who care in the best position to support us. In Neil Smith’s “Green Fluorescent Protein,” When Max was fighting with Ruby-Doo; he wants to stay with his mom for comfort. GFP is a metaphor for Max’s realization that he is queer. Max is ensuring of his feelings towards Ruby-doo. “In Pop’s arms, the white guinea pig turns a brilliant green…… It’s very weird and really scary. [it’s] kind of beautiful in an unexpected way” [50]. He tried to say same sex love can be weird and scary, but it’s love and love it a beautiful thing. Family plays an important role in max’s life. This is what for example Max’s said “I want to say, don’t go. Not that I’m afraid her date

Based on the samples the expression of rGFP was the greatest in the E3 both quantitatively and qualitatively. The purity of rGFP was however low. The rGFP protein was purified using the Ni+2 agarose chromatography. This part of the experiment did not achieve a high purity. Causes of this may have incorrect pipetting mechanism or incorrect amount deposition. Improving pipetting skills might have lead to more accurate results. A better comprehension of the purpose of each step in the protocol may have resulted in improved results. Also personally having to use GCE of a partner’s could have altered the results. Being more strict with the lab procedures, and not messing up with the timings of certain steps by accident would have improved data. Although minor errors may have occurred the greatest fluorescence occurred in wash fraction W4 and elution fraction E3 quantitatively, and this data matched the qualitative observation of the fluorescence in both these samples. W4 had the greatest protein amount. W1 was the void volume and only contained breaking buffer without rGFP therefore there should have been no fluorescence, and this occurred correctly. Samples W2 and W3 had slight fluorescence which could be explained by the rGFP lacking the His6 tag required to bind to the Ni+2 agarose, or all the binding sites in the column could

Ps: the iodine was already really dark so it was very hard to see much difference between the control and the others.

-terminal fragment of the YFP was fused to the N-terminus of TuHC and AtCA1 to make YFPN-TuHC and YFPN-AtCA1, respectively, while the C-terminal fragment of the YFP was fused to the N-terminus of TuHC and TuMV CP (TuCP)

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).

The association of FMRP with ribosomes is mRNA dependent via large ribonucleoprotein (RNP) particles. RNP particles contain other proteins including FXR1P, FXR2P, YB-1, CYFIP1, CYF1P2, NUFIP1 and nucleolin.

As most, nuclear receptors including retinoid receptors possess similarities in their structural organization. Based on homology with other nuclear receptors, RAR and RXR receptors consists six conserved regions known as A–F. The N terminal region contains two domains (A/B) are designated as AF1 and they are the least conserved domains and highly variable in length which exhibit a ligand-independent transactivation activity. So far, there is no three dimensional structure has been predicted for A/B domain. (9).

To understand the transformation lab we did, you need some background information to help understand what we did. The DNA structure is formed in a double helix which means it has two strands and consists of nucleotides. Each nucleotide contains deoxyribose sugars that are bonded by phosphodiester which bond to a phosphate group and a nitrogen base. The nitrogen base matches up to the nitrogen base on the opposite strand of the double helix. There are two types of nitrogen bases that occur, purine which is either A or G which form a hydrogen bond with pyrimidine which is either T or C. When DNA is replicated the hydrogen bonds that hold the strands together break down by an enzyme and then the RNA primase is added so DNA polymerase 3 can attach

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

In the past scientist have used the bacteria Escherichia coli (E. coli) in Synthetic Biology to allow the bacterial species to recognise and respond to light frequencies providing greater knowledge of complex biological systems (Levskaya et al. 2005). E. coli is a suitable chassis to express Green Fluorescent Protein (GFP) as the bacteria’s plasmid helps to establish GFP (Chavshin et al. 2013). The bacterial plasmid is a maltose-binding protein that helps E. coli and GFP fuse together and allows for detection of gene expression and controlling internal environments (Feilmeier et al. 2000). As GFP is a useful biological indicator due to its fluorescent structure (Mazzola et al. 2005) and through the ability of E. coli responding to light, GFP is easily expressed through the process of light stimulation (Lee, J et al. 2013). Through the unique structure and functions found within E. coli, it allows for successful expression of GFP making E. coli a suitable base organism.

Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the

Polyacrylamide gels are usually preferred over other gel types such as agarose in protein electrophoresis because of various benefits such as its ability to be cast in various pore sizes which provides options for the researchers who observe protein samples with various Kda’s depending on how separated they aim the bands to be or how well the sample is sieved. It is also preferred because of its charge neutrality upon setting as it does not interfere with the electrophoresis which is highly reliant on the sample and the surrounding’s charges within the system. Lastly, the gel itself is colorless and protein stains do not adhere to the gel so there will be minimal problems with unnecessary stains that aren’t from the staining solution.

Determination of concentration of crude extract was based on Bradford Coomassie brilliant blue assay which forms a complex between the protein dye and protein in solution which causes a shift in the maximum absorption of light that passes the dye from 465 to 595 nm (Bradford, 1976). The amount of light that passes through the protein solution is

Present study is based on laboratory work; however the plant material for experimental purpose will be collected from experimental farm of CSIR-IIIM (32.73N and 74.87E).