Separation and Identification of Bovine Milk α-lactalbumin and β-lactoglobulin Corbin Croom, Tyler Donathan, Cama Dooley, Nicholas Johnson October 15, 2014 Abstract Analytical gel electrophoresis has been used to separate and identify many kinds of proteins including α-lactalbumin and β-lactoglobulin. Both proteins can be derived from bovine milk. Skim milk was centrifuged with a variance in pH to precipitate casein proteins. Whey material was filtered and eluted through a Sephadex G-50 column. UV Spectrophotometric analysis confirmed the presence of the protein within the fractions. Separation and identification of α-lactalbumin and β-lactoglobulin was determined with SDS-polyacrylimide gel electrophoresis. With the molecular …show more content…
The supernatant was next decanted into a small beaker, while the lipid layer pellet was kept in the bottom of the beaker. The pH of this supernatant was 4.51. For this process, 12 M acetic acid was added to the supernatant by dropwise addition, followed by the dropwise addition of 0.5 M HCl. The solution was then heated with stirring at a temperature between 35 and 40 degrees Celsius for 30 min. The solution was next decanted into a centrifuge tube and centrifuged for 30 min. The whey was collected by filtering through a 0.45 micrometer syringe cartridge filter and stored in the freezer. Sephadex chromatography was prepared using dry beads swollen in water along with a Tris buffer to equilibrate the gel. The 4.0 mL of whey was measured to apply to the column. After the solvent had just drained through the top of the sephandex column, α-lactalbumin solution was added dropwise to the top of the gel through a pipet. After the solution entered the gel, approximately 10 mL of buffer was added to the top of the column. The stopper was placed on top of the column and the eluting solvent supply allowed continual rate of buffer into the column. The flow rate out of the column was adjusted to 2 mL/48 sec. 2 ml fractions were collected in 59 test tubes in a fraction collector. 0.5 mL of Tris buffer was added to each fraction as a dilution. The test tube fractions were transferred …show more content…
fraction number. These peaks occurred at fraction number 43 and fraction number 50, with absorbances close to 1. Around these fraction numbers was where the largest elution of protein and likely α-lactalbumin was present. Due to high amino acid content of phenylalanine, tryptophan, and tyrosine, absorbance occurs at 280 nm. The high 280 nm absorbance would suggest a higher amount of protein elution in the fraction. The early fraction numbers had significantly high absorbance between fractions 4 and 9. This was due to quick mobility of the protein through the sephadex column during the size exclusion chromatography before fractions were gathered. The absorbance of the protein fragments: 7, 8, and 9 were all between 0.59 and 0.62. Protein fragments with absorbance values above 0.5 had a noticeable amount of desirable protein within the fraction. The electrophoresis gel was labeled in comparison with a pre-stained protein ladder as shown in Figure 2. The 10 kDa served as a base for the accuracy of the other protein ladder measurements. The 17 kDa measurement on the protein ladder was compared with the band values of the protein fractions. α-lactalbumin has a molecular weight of 14.2 kDa. The second protein band that eluted from the fractions in the gel was significantly close to 14 kDa in comparison with the 17 kDa measurement, revealing that α-lactalbumin was present in the fractions. β-lactoglobulin
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