The objective of the study was to determine if there was an interaction between USP7 and EBNA1. Prior to the experiments, the DNA sequences of EBNA1 and its fusion tag—GST, USP7 and its corresponding fusion tag—6xHis, and the GST tag alone were each inserted into three distinct 10mL cultures of E.coli BL21 using plasmids. Consequently, each culture expressed one of 6xHis-USP7, GST, and GST-EBNA1 peptide. The GST-EBNA1 culture was placed in 45mL of LB Amp+ medium, whereas the 6xHis-USP7 and GST were mixed with 45mL of LB Amp+ Km+ medium. The cultures were left in in shaking incubators at 250rpm for an hour at 37°C. An optical density reading was taken of 1mL samples of each of the cultures. The optical densities fell within the ideal range of 0.8 to 1.0, indicating that sufficient protein had been expressed and no further incubation was needed. 150µL of GST-EBNA1 was taken from the cuvette and briefly centrifuged; the pellets were isolated at -20°C. Next, 0.55mL of 0.1M IPTG was added to each culture and placed in the shaking incubator at 37°C for one hour to prompt the expression of peptides. The induced cultures were centrifuged at 4°C for 20minutes at 4000rpm, and the pellets were stored at -20°C until use. Lysis/Binding buffer was made with 5µL of 5M imidazole and 5mL of Common buffer (500mM NaCl and 50mM Tris-HCL at pH7.5) and left on ice. 1mL of this buffer was added to thawed 6xHis-USP7 pellets. The mixture was vortexed, placed on ice, and lysed with an enclosed sonicator for four 20 second pulses at 15 second intervals, at 30% intensity. The refrigerated micro-centrifuge was used for 10 minutes at 13000rpm to isolate insoluble proteins as well as un-lysed cells. The supernatant was isolated, and 10µL of the lysate was s... ... middle of paper ... ...xHis-USP7 washed through the GST column and failed to bind to the GST protein, yielding a single band on the SDS-PAGE. The 6xHis-USP7 band observed is extremely faint, likely due to the fact that the volume of the purified protein used was only 20µL. Nevertheless, the 6xHis-USP7 did not wash through the GST-EBNA1 column, and the GST-EBNA1 pull-down unbound protein sample yielded no bands (Figure 4). Moreover, neither the GST pull-down wash nor the GST-EBNA1 pull-down wash yielded any bands. Upon washing the column with GST elution buffer rich in reduced glutathione, the GST pull-down eluent yielded a single thick band corresponding to the GST protein. The GST-EBNA1 pull-down elution, however, yielded two distinct bands. The heavier band that travelled a shorter distance corresponded to GST-EBNA1, while the lighter band corresponded to 6xHis-USP7 (Figure 4).
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things. In this experiment, the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO or +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin.
Moreover, the sensitivity and specificity of the western blot (Immunoblotting) enables it a common technique for determining specific protein levels in clinical samples. Since the antibody is specific to the antigen immunospecificity it enables the target protein to be identified. Western blotting can produce quantitative data about that protein, which in this case shows the difference between bands in each of the protein samples. The western blot is an analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. The proteins are then transferred to a membrane (in this case, nitrocellulose), where they are stained with antibodies specific to the target protein [1] [2].
Ligation one was a 1:1 molar ratio pET-41a (+) vector: egfp insert that used 50ng NotI/NcoI cut pET-41a (+) DNA, 7ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Ligation two was a 1:3 molar ratio pET-41a (+) vector: egfp insert made up of 50ng NcoI/NotI cut pET-41a (+), 21ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Water was sterilized and deionized. The remaining three ligation samples served as controls. Ligation three contained 57ng uncut pET-41a (+)/EGFP recombinant plasmid DNA and sterile water. Ligation 4 was a negative control that consisted of only sterile water. Ligation five lacks DNA ligase but has the same properties of the 1:3 molar ratio pET-41a (+)/EGFP vector.
Wissmueller S., Font J., Liew C.W., Cram E., Schroeder T., Turner J., Crossley M., Mackay J.P. and Matthews J.M. (2011). Protein-protein interactions: analysis of a false positive GST pulldown result. Proteins. 79 (8), pp. 2365-2371.
These six samples (crude -/+, broken -/+, and whole -/+) were spun at 5000 rpm, and the resulting pellets were isolated and resuspended in DNase buffer. The set of suspensions labeled with a (+) was incubated in DNase enzyme for 15 minutes, and afterwards incubated in 15 uL of STOP solution. All six samples were lysed for DNA extraction with DNA extraction buffer, and micro-centrifuged at maximum speed. To precipitate the extracted DNA, the supernatants from each of the six samples were added to their correspondingly labeled micro-centrifuge tubes containing 7% ethanol (Parent et. al, 2008To bind the DNA, the ethanol lysate mixtures were transferred to labeled spin columns and spun for one minute in the micro-centrifuge at maximum speed. To wash the bound DNA, the spin columns were washed and spun three times at maximum speed. In order to elute the bound DNA, the samples were washed in 80 uL of distilled water and spun again for 2 minutes at maximum speed (Parent et. al,
Sansone, Randy A., and Lori A. Sansone. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 09 Apr. 2014.
CP consists of a single domain with high α-helical content [4]. The N-terminal part this domain is surface exposed whereas the C-terminal region buried in the virion. Several experiments indicate the CP is an O-glycoprotein. Equal amounts of galactose and fructose residues are O-linked to an acetylated serine residue at the N-terminal region [2]. This mediates the formation of a structured...
Schulman, Joshua M., and David E. Fisher. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 28 Aug. 0005. Web. 24 Apr. 2014.
The synthetic A and B chains are then inserted into the bacteria’s gene for B-galactosidase, which is carried in the vectors plasmid. The vector for the production of insulin is a weakened strain of the common bacteria Escherichia coli, usually called E. coli. The recombinant plasmids are then reintroduced to the E. coli cells. As the B-galactosidase replicates in a cell undergoing mitosis the insulin gene is expressed. To yield substantial amounts of insulin millions of the bacteria possessing the recombinant plasmid are required.
In To Kill a Mockingbird by Harper Lee, Atticus Finch says, "Shoot all the bluejays you want, if you can hit em', but remember it's a sin To Kill a Mockingbird." This quote demonstrates Atticus's firm belioef in the Golden Rule. The Golden Rule is to treat others how you want to be treated. Atticus follows the Golden Rule throughout the novel, including while defending Tom Robinson, a local African American accused of raping Mayella Ewell. Due to Atticus's firm belief in the Golden Rule and the fact he must live with himself before others and tries to teach his children the right thing to do, it makes sense for him to defend Tom Robinson.
"The Species of the Secondary Protein Structure. Virtual Chembook - Elmhurst College. Retrieved July 25, 2008, from http://www.cd http://www.elmhurst.edu/chm/vchembook/566secprotein.html Silk Road Foundation. n.d. - n.d. - n.d.
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.
If we examine the detailed structures of many transmembrane proteins, we see that they often have three different domains, two hydrophilic and one hydrophobic .(fig 1&2) A hydrophilic domain (consisting of hydrophilic amino acids) at the N-terminus pokes out in the extracellular medium, a hydrophobic domain in the middle of the amino acid chain, often only 20-30 amino acids long, is threaded through the plasma membrane, and a hydrophilic domain at the C-terminus protrudes into the cytoplasm. The transmembrane domain, because it is made of amino acids having hydrophobic side chains, exists comfortably in the hydrophobic inner layers of the plasma membrane. Because these transmembrane domains anchor many proteins in the lipid bilayer, these proteins are not free-floating and cannot be isolated and purified biochemically without first dissolving away the lipid bilayer with detergents. (Indeed, much of the washing we do in our lives is necessitated by the need to solubilize proteins that are embedded in lipid membranes using detergents!)