Lactoglobulin Lab Report

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.

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

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.

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.

Protein standards were previously loaded into the first rows of the well plate of which the concentrations were as followed; 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, and 0 μg/mL (PBS only). After making note of the rows used the plates were loaded onto the selected protein row, followed by the addition of 200 μL of Bradford reagent to each well using a multichannel pipette. The absorbance of the plate was read at 595nm and recorded. The NT-2 and LPS-2 protein sample tubes were labeled for easy identification in the following lab and the protein samples were stored in the freezer for 1 week.

Standard curve created from the BSA standards results. The equation derived from the standard curve helped calculate the protein concentration of GFP. Figure 4. Comparing the Western blot results to the protein ladder. (a) Shows the protein ladder, (b) is the ladder obtained from the gel electrophoresis, and (c) shows two bands from the Western

Tube three contained 1.0 mL of Buffer, pH 6.0 0.1 M NaPO4 including 0.4 mL of 0.006 M catechol, with 600 uM of substrate concentration and 1.6 mL of distilled water. Tube four contained 1.0 mL of Buffer, pH 6.0 0.1 M NaPO4 including 0.8 mL of 0.006 M catechol, with 1200 uM of substrate concentration and 1.2 mL of distilled water. And finally, Tube five contained 1.0 mL of Buffer, pH 6.0 0.1 M NaPO4 including 1.6 mL of 0.006 M catechol, with 2400 uM of substrate concentration and 0.4 mL of distilled water. All five of the test tubes also contained 1.0 mL of enzyme, which would be added right before testing each tube for absorbance. After preparing all of the test tubes, each tube was inserted in the spectrophotometer one-by-one and recorded its change in absorbance for five minutes.

Next, the biuret test for protein turned solutions in test tube 9 and 12 purple, the biuret test was positive, which proves that protein was present in the solutions, whereas test tubes 1,2,3,4,5,6,7,8,10, and 11 had negative results, this is because those solutions do not have peptide bonds and a peptide bond is necessary for the biuret test to be positive. An unknown was also tested in this experiment and we have concluded that the unknown #267 is a protein because it

The chicken breast muscle had the highest number of proteins present (13), while the porcine kidney had the least (3) amount present (Table 2). The protein with the largest mass appeared to be the first protein in the chicken breast muscle lane at 192.5 kDa (Table 2). There was also a trend that two protein bands with an apparent mass of 64.6 ± 2.67 kDa and 78.5 ± 2.13 kDa appeared in each of the different acetone powder lanes (Table 2). The protein with the smallest mass was found in the calf liver at 17.0 kDa (Table

Protein samples separated by size via SDS-Page can be identified using mass spectroscopy; which will require proteins from the gel to be treated using trypsin; which is an enzyme that digests proteins. Assuming that samples were treated and storaged properly, the digestion of peptides begins with the rehydration of proteins using trypsin, followed by the incubation of proteins using a

The testing of various proteins was performed by comparing the molecular weight of proteins using SDS PAGE. The molecular focus in the lab was the testing of proteins, which are macromolecules consisting of amino acid monomers linked through chemical bonds. These proteins have a hierarchy of structure that consists of folding that determines the direct function of each protein.. The molecular weight of these proteins were measured using SDS PAGE. SDS PAGE stands for sodium dodecylsulfate polyacrylamide gel electrophoresis. SDS is an anion detergent that binds with the protein structures and causes them to separate due to the change in bonding charge. SDS and heat are how the proteins are denatured. The process of denaturing a protein is breaking

After purifying the proteins, the students will be able to compare the molecular weights of the samples by running a denatured SDS polyacrylamide gel (Purification & Size Determination of GFP & BFP, EDVOTEK). This will show students which proteins different from one another and how denaturing can affect the proteins. Background:

In test tube D add two millilitres of the sucrose solution and one millilitre of enzyme solution. 10. Repeat step 4. 11.

The purpose of this lab was to purify and test a GFP protein via several laboratory methods for the purpose of purifying and testing the protein in SDS-PAGE. To purify the protein chromatography and gel electrophoresis were the methods used in the experiment. GFP in the samples were tested using an ultraviolet light. When GFP was found present the cell were transformed into a petri dish containing ampicillin and arabinose. The cells were then lysed and SDS-PAGE was used to test.

Different techniques and principles for protein extraction and characterization were demonstrated in this experiment. Various proteins were extracted from different sources: 1.67 g yeast invertase, 1.03 g egg white albumin, and 5.15 g of milk casein. Activity assay for invertase was performed using Benedict’s test and the enzymes inverting action on sucrose was confirmed. Warburg-Christian Method and Bradford Assay were also employed to determine the protein concentration in the albumin and the casein samples. The concentrations for the albumin and casein samples were found to be 0.519 and 0.327 mg/mL, respectively based on Warburg-Christian Assay; and 6.5x10-3¬ and 1.9x10-2 mg/mL