Binary Chromatography Lab Report

In Figure 3, there is a collected data with the total washes and the elution fraction with the RFUs from the Ni+2 Agarose column. E3 had the highest elution fraction with 8554 RFUs. In the Bradford assay, E3 had the highest protein amount with 65.5ug. The specific activity for E3 was about 1.28E5 RFUs/mg. Overall, in the Figure 3, the E1-E6 had most of the rGFPs due to being bonded in Ni+2. W1-W6 rGFP is much smaller than the E1-E6 amounts.

The investigation is showing how enzymes work inside a mammal's stomach. Rennin is the enzyme found in young mammals and has more effect

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

In the Affinity Chromatography experiment we were purifying our Con A proteins. In general, affinity chromatography is a technique that is used for isolating a protein, in our case Con A from a large amount of other macromolecules. Our protein of interest is captured using a microbead matrix while we let everything else flow through the column. The Sephadex matrix is made of cross-linked glucose or dextran and because our Con A has an affinity for glucose it is able to bind to those beads. In general, we began by equilibrating our column with NaCl, then poured Jack Bean Meal Extract which so happens to contain Con A through our column, the Con A then binded to the Sephadex beads, and finally we eluded with a dextrose solution so that

P1 and P2 centrifuged for three minutes at 1000rpm. Supernatant transferred to Eppendorf tubes, 1ml of each saved and set aside. P1 diluted by a factor of 100 and loaded in a column with 5mL. 5mL undiluted P2 loaded into a separate column. 10mL Buffer A used to wash the column. A 10mL of low-salt buffer loaded into each column, 1-2mL collected into each cuvette. Cuvettes scanned with a spectrophotometer, blanked with low salt buffer. Fraction contained the most protein identified and isolated into an Eppendorf tube and placed on ice. The same procedure followed for medium salt and high salt, the blank correlated with loaded buffer. The beads cleaned with a 10mL resin cleaning buffer.

Lab 8: Investigation 5 Lab question: How do you separate molecules that are attracted to one another? Procedure: Place a small amount of solvent in separate beakers. Then place the mixture on chromatography paper and put the paper in each beaker. Controls:

The reagent used to transfect pCas9-GFP into 293 cells was polyethylenimine (PEI). PEI is a cationic polymer and is the transfecting agent used to introduce pCas9-GFP into the 293 cells via endocytosis and thus releasing the DNA into the nucleus. OptiMEM was also used for the transfection of the cells; it provided a biological environment to the eukaryotic cells, thus increasing the rate of transfection. Following the

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)

This experiment will help students to get familiar with Ion Exchange Chromatography (IEC) which is used mostly to separate unknown mixture of metal ions. Anion and cation are known as two types of IEC. This column chromatography contains two phase such as stationary phase and mobile phase. In this experiment, stationary phase usually is made of organic resin or polymers because these are insoluble in water and barely inert. AG 1-X8 resin is used in this experiment because of their strongly basic anion exchanger. 3M HCl is used as mobile phase in this column chromatography. Cobalt (II) and Copper(II) bounds to different number of Cl- ligands, so they have different affinity to the resin.

For this experiment, Gas Chromatography (GC) is utilized to observe the streo- and regiochemistry among four elimination products under either basic or acidic conditions. The reagents used in this experiment include primary and secondary alcohols which are in acidic conditions and dehydrobromintion of primary and secondary alkyl halides in basic conditions. When reacted the gas products are collected and undergo further analysis in the GC which will separate the product into its components and will show the different distributions among the product. Theory

Two methods were utilized in order to purify and extract the pigmented compounds within MI-1 and MI-2. These methods were column chromatography with a silica base gel and solid phase extraction utilizing LC-Diol and LC-Si cartridges. These methods all revolve around trying to purify polar compounds from nonpolar impurities and were chosen since prodigiosin is a polar compound containing three pyrrole rings (6). Upon completion of techniques, two primary compounds were extracted via column chromatography: a red pigmented compound, and a UV sensitive compound. The UV sensitive compound was stored as it was not the focus of this study. The LC-Diol cartridge successful in extracting the red pigmented compound, while the LC-Si cartridge would no elute the compound.

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

Structure and mechanism of alkaline phosphatase. Annu Rev Biophys Biomol Struct. 1992; 21:441-83. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1525473

Chromatography Investigation Chromatography is a highly regarded technique used to separate the components of a mixture. It is based on the principle that each component possesses a unique affinity for a stationary phase and a mobile phase. The components that are more inclined to enter the mobile phase will migrate further on the chromatogram and distinguish themselves from the other components. The type of solvent used in chromatography is known to directly affect the separation of the mixture. In this experiment, thin-layer and column chromatography will be utilized to separate the numerous chlorophyll and carotenoid pigments of a spinach extract.

Gel-Filtration Chromatography is a commonly used method used in order purify a protein from a mixture, by means of separations. Different biomolecules differ in size, or their molecular weight. In the gel matrix inside the chromatography column, there are gel beads which are porous to allow certain sized molecules to enter. The molecules that are able to enter the pores of the gel, are held in stationary phase and will elute from the column later on, these are usually smaller, to medium sized molecules. Larger molecules that are not able to fit in the pores will elute out of the column first, they are involved in mobile phase where they just go straight through the column without interacting with the gel beads. Smaller molecules will have a higher elution volume, while the larger molecules will have a lower elution volume. The volume to elute the protein is inversely proportional to the molecules size.