Lab Report

The filter flask, vacuum, and SNAP i.d. 2.0 were securely connected with tubing. The proteins were transferred to a membrane and was incubated with primary antibody. The primary antibody was then washed three times. Afterwards a secondary antibody was added and the same procedure was repeated. 10mL of 5-bromo-4-chloro-3-indoyl phosphate/ nitro blue tetrazolium chloride chromogenic substrate solution was added to the blot which was then wrapped with aluminum foil and labeled.

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).

This was done by stacking a sponge, and filter paper behind a gel with nitrocellulose, with the two gels facing each other, then was all compressed between a locking cassette lids. Once transferred the bands were stained with for 2 minutes with 20 ml of Ponceau S then rinsed with distilled water until the bands appear just right. Next, the membranes were placed back to back with protein facing outward from eachother and was incubated for 30 minutes in 30 ml of 5% non fat dry milk/TBS. after the 30 ml was discarded, 7ml of the Primary antibody, mouse IgG anti Xpress epitope MAb solution was added and incubated with the shaking platform for 45 minutes. Then in triplicate the membrane was washed with 30 ml of 0.05% tween20/TBS and shaking for 5 minutes. 7ml of a secondary antibody, (sheep IgG anti-mouse IgG conjugated with horse radish peroxidase polycolonal antiserum), (antibody diluted 1:1,500) was added and incubated on a shaking platform for 45 minutes. Then perform the wash steps again in triplicate, but this time do a fourth final wash with TBS and incubate on shaking platform for 5 minutes like the previous washes. Finally The membrane was developed by the addition of TMB, and rocking the solution back and forth until the bands were apparent, then the membrane was put into water to stop he reaction, token out dried and the results were

Samples P1 and P2 were used in this part of the lab. The beads were positively charged while the proteins were negatively charged. There were a total of 3 different salt solution concentrations, low salt, medium salt, and high salt. The salt concentration was increased in order to elute the molecules that were attached to the column. The weakest were washed away first while the strongest was the last.

In this experiment, two sets of variables were manipulated to see the effect it would have on the crystallization of the lysozymes via the hanging drop method. In order to help visualize which will bring optimal crystallization of the protein, two sets of variables were tested in this experiment: salt concentration and the concentration of polyethylene glycol (PEG). Our experiment dealt with increasing the concentration of both these sets of variables to see how it would affect the quantity of protein crystals produced. As concentration of NaCl increased, so did the yield of crystals and the same could be said with PEG. As PEG concentration neared a concentration of 20% we saw an increase of crystals.

The starting amount of synthetic mRNA was 100 nmol but was split into two Eppendorf tubes of 50 nmol each, with 5 nmol of spin label for each tube. Since the R5 spin labels are hydroscopic and light sensitive, they were purged with nitrogen gas and foiled. To dissolve the R5 spin labels, a solution made with 100 µL of acetone and 15 mg of sodium iodide was also purged with nitrogen gas and vortexed with the R5 spin labels. The two tubes of mRNA were mixed with 20 µL of purged acetonitrile, 22.4 µL of autoclaved water, and 200 µL of TE (Tris-EDTA) buffer. The R5 mixtures were then combined and vortexed with the mRNA solutions. After centrifuging the two combined R5 and mRNA solutions at 18000g for 10 minutes, they were mixed well by pipetting up and down the solutions. The two resulting solutions were foiled and placed on a spinning rotor at

Through a series of experiments we are trying to demonstrate that we are able to express and purify a recombinant GFP in E. coli strain BL21(DE3)<pRSETA-GFPUV using a Ni2+ agarose affinity chromatography method. The findings showed that one the highest amount of activity came from elution 3 with 15144 RFU’s. The estimated protein amount in Elution 3 was about 49ug using the Bradford assay, and specific activity of 309061 RFU/mg, the SDS page showed that E3s purity was about 33 %. The SDS Page showed faint bands at both, 28kDa and 32 kDa, and western Blot analysis, showed that there was definite rGFP found in the E3, as well as all the Fractions.

Described by Coligan et al (2005), Glutaraldehyde coupling was needed to conjugate the two peptides individually to bovine serum albumin. BSA dissolved in 500ul of 0.1M borate buffer pH10 was manipulated to mix with the air-oxidized peptide solution that was already prepared taking note that the mixture has to be on a 1:10peptide-BSA ratio. Moreover, until a 1:10:1 peptide-BSA-glutaraldehyde ratio was achieved, the peptide-BSA mixture was combined with 1% glutaraldehyde. Appendix B includes the computations for the preparation of antigen conjugation. After, the mixture was incubated for 2 hours at room temperature and was diluted with distilled water until a 1mg of peptide in 1ml of the peptide-BSA-glutaraldehyde

Ni+2 affinity chromatography purifies 6xHis USP7 fusion protein by using a Ni2+ resin and an imidazole ligand to elute the desired protein; in this method 6-9 histidine residues are used as the tag. Imidazole was used in many different concentrations to disrupt the interaction between the His residues and NI+2 also, higher concentrations are needed to elute which allows imidazole to compete for binding to the resin with the His-tag. However, GST affinity chromatography purifies GST and GST-EBNA. This method allows for the elution of GST by using a GSH resin and ligand. It should also be noted that the GST tag acts as a substrate for the ligand to bind to immobilize the affinity column(Bornhorst and Falke, 2004).

Before starting this type of experiment, it is important to have an antibody that knows the protein that will be worked with. The experiment used in this article used lysates of cultured mammalian cells as well as purified bait proteins. To have

Protein properties are directly affected by their environment, which depends on pH, temperature, solvent effects, and pressure. With these affects has come more desire for better techniques in the purification and separation processes. These processes hopefully will prevent loss of biological activity and denaturation. With ion exchange chromatography comes many advantages over column chromatography such as union between the target protein and functional groups at a very quick rate, exponential growth without difficulty, short processing times, no heating, no extreme pH range, and a multitude of other things (Santos et al. 2012). Ion exchange chromatography in short is ideal due to the low cost, longevity through harsh cleanings, and high

Figure 1: Antibody model showing subunit composition and domain distribution along the polypeptide chains. Single-chain fragment variable (scFv) antibody generated by recombinant antibody technology appears in the shaded area. [2].

Objective: The purpose of today’s laboratory is to understand how size exclusion chromatography plays a role in purifying protein which is an important concept to understand as it helps answer the mysteries which lay behind proteins such as the structure or the enzyme kinetics. The fractions which were collected were used to analyze the amount of target protein in each sample.

• Site-specific targeting can be achieved by attaching targeting ligands to surface of particlesor use of magnetic

The data obtained from DSC provides important information on proteins stability during processes of development, multiplication and also in formulation of drug candidates.