Bovine Serum Albumin

The specificity of albumin binding experiment was to determine the binding interactions that occur between serum albumin and three synthetic dyes with the use of electrophoretic procedure. Whole blood, or plasma. Clots upon standing and if the clot is removed, the remaining straw colored fluid is called serum. The major protein in serum is albumin which functions as a carrier molecule for the transport of certain small molecular weight compounds in blood. Molecules that bind to serum albumin are fatty acids, hormones and some synthetic dyes. In this experiment the synthetic dyes used are Bromophenol Blue, Ponceau S and Orange G. we observed that free dyes not bound to albumin migrate faster that albumin or dyes bound to albumin. This

The IgG antibody was separated by using AKTA FPLC by setting the method as:- Column- HiTrap protein G HP 1 mL; Column pressure limit- 0.300 MPa; Flow rate- 1 mL/minute; Sample injection- 0.200 mL; Loading buffer, A- 0.05 M sodium phosphate, 0.15M NaCl, pH 7.0; Elution buffer, B- 0.1 M glycine-HCl, pH 2.7; Neutralization buffer, C- 1 M Tris-HCl, pH 9.0. Different column volume for individual step was then set as- 1 CV 0% B wash and equilibration step; 1 CV 0% B loading; 4 CV 0% B washing; 10 CV 100% B eluting; 5 CV 0% B equilibration. After setting the method, the system was washed with buffer B at the flow rate of

Quantification of proteins is needed to determine the progress of protein purification. As the protein becomes more purified, its specific activity will increase as well. In Experiment 4.1, dilutions of PNP are prepared, and the Bradford Method was used to measure protein concentration.

A glass column with inner diameter of 1 cm, was packed with Bio-Gel P-100 (BIO-RAD) beads suspended in equilibrium buffer of 20 mM phosphate buffer with a pH of 7.0 - 7.4. The height of the total content was kept at 5cm. The sample mix with total volume of .1ml contained 2 mg/ml Blue Dextran (blue, 2 MDa), 5 µl yellow food coloring (yellow, ~500 Da), 2 mg/ml Hemoglobin (red), 2 mg/ml of BSA in phosphate buffer. As soon as the sample mix was added to the column, 0.25 ml (~2 drops) of fractions were collected in the Eppendorf tubes until all the colored bands eluted the column because it was assumed that the colored band represented protein content. Approximately 1 µl of each of the fractions collected were spotted on to nitrocellulose filter using capillary pipet. After the filter was dried, it was incubated in 50% methanol for 5 minutes then it was incubated in Amido Black (0.1% amido black 10B in 30% methanol) for 5 minutes. Next, the filter was

The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique used to measure the concentration of an analyte (usually antibodies or antigens) in solution. In the practical anti-BSA antibodies that had undergone serial dilutions were added to a BSA solution in an ELISA plate with goal of seeing how the concentration of anti-BSA antibodies would affect the colour change of the BSA solution. The results clearly showed a direct correlation as the more diluted the anti-BSA antibody solutions became the lower the Wavelength readings at 405nm, which showed that there was less of a colour change.

For this experiment, whole bovine blood was used. The first process was to separate the blood into cellular and plasma fractions. 100 µL of whole bovine blood was transferred into a yellow microcentrifuge tube that was labeled WB using a P-200. 50 µL of whole blood was added to a blue microcentrifuge tube labeled WB. Both tubes were capped and placed in ice. 2 mL of the remaining blood was transferred into a Clear 2 mL tube using a P-2000 and centrifuged for 5 minutes at 8000 RPM. Afterward, 800 µL of the supernatant from the Clear tube was transferred into a yellow tube labeled WP and 50 µL of the supernatant was added to a blue tube labeled WP. These tubes were then capped and kept on ice.

A clarified and physiologic buffered (pH 7 to 8) is added to the antibodies and purified ZA2GP to form a mixture that is added to the immobilized ligand.

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

The tray lid was discarded and the dish containing membrane was put on the rocking platform for 15 minutes, washing the blot three times with 10 mL of TBS Tween at 5-minute intervals. Following the removal of all liquid from the dish at the end of the wash 10 mL of secondary antibody (goat anti-rabbit IgG HRP) diluted in milk blocking solution were added. Afterwards, the blot was incubated on the rocking platform for 20 minutes. During this incubation period the coomassie blue stained gel was placed on white paper for clear visibility and a photo was taken to document its appearance. This gel was previously incubated in Coomassie blue for approximately 1 hour. The gel was then discarded in the biohazard bag. After removing the membrane from the rocking platform the secondary antibody solution was removed from the membrane and discarded. Then 10 mL of TBS-Tween were added to the blot and the tray was manually shaken back and forth to remove leftover milk blocking solution and secondary antibody, which was immediately poured off. The blot was washed three times with 10 mL of TBS-Tween, at 5-minute intervals using the rocking platform. Upon completion of the washes a picture of the membrane was taken. All liquid was removed

Once the experiment is carried out on the tissue for use of staining and the antibodies are not coming up with any reactivity involvement with the antigen then the antigen retrieval methods are used before staining the fixed tissue to enable the antibodies to work better. In this experiment method it helps to use the antigen retrieval method because the methylene bridges that are formed in the fixation step has caused a cross linking on the tissue sample from the proteins and because of this it has caused a mask effect to occur on the protein epitope that the antibody used would have usually detected. Antigen retrieval helps by causing an unfold in the protein on the tissue of the rat pancreas or what other tissue is being analysed and because it has caused this reversal and unfold in the protein it now allows the antibody to detect the protein and bind to

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).

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.

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

They only target part of the pathogen to provoke an immune response. This may be done by isolating a specific protein from a pathogen and presenting it as an antigen on its own. These vaccines can be created by genetic engineering as well, via a gene coding. A gene coding for a vaccine protein is inserted into another virus, when the carrier virus reproduces or when the producer cell metabolizes the vaccine protein is created as a product. The end result is a recombinant vaccine. Conjugate vaccines are made using pieces from the coats of bacteria. They are a combination of two different components. The coats are chemically linked to a carrier protein and combination is used as a vaccine. These vaccines are used to create more powerful combined immune

This protein is taken with the help of the injection. From the container, the intake quantity is needed to inject into the injection