Sodium dodecyl sulfate polyacrylamide gel electrophoresis also known as SDS-PAGE is one of the methods for determining the molecular weight of unknown proteins. SDS is an anionic molecule which denaturizes proteins and brings it back to its’ primary structure and it also provides a negative charge to the uncharged molecule. The SDS-PAGE enables the separation of proteins based on their sizes. The larger the size of the protein, the harder it is to travel through the gel thus heavier proteins stay near the cathode side of the gel. For this experiment, a software named Gel Analyzer was used in order to obtain the molecular weight of the unknown proteins with the help of a protein ladder with known molecular weight and protein concentration.
In the first stage in the normal body, vitamin B12 binding proteins that found in saliva to enters the stomach. Then the stomach uses hydrochloric acid to liberate vitamin B12 from the proteins. However, in the first stage of pernicious anemia, the stomach excretes a small amount of hydrochloric acid. This leads to a failure of separate vitamin B12 from food proteins, which inhibit vitamin B12 from absorption. In addition, the lack of secretion of hydrochloric acid provides a suitable environment for a reproduction of gut bacteria.
The technique is often used in research to detect specific proteins which have extracted from cells. In this process, a mixture of proteins separated based on two distinguishing properties which are molecular weight and antibody binding specificity. According to the procedure, proteins first separated based on size which have to perform with SDS-PAGE. Next, the proteins from the gel are then transferred to a polymer membrane (PVDF or nitrocellulose) to make them more accessible to the antibodies that specific to the target protein. Cytotoxic assay Cytotoxicity is described as the quality of being toxic to cells.
TLC was used to identify the actual unknown product as well as other products/reactants present in the filtered solution. The procedure was conducted by placing a TLC plate in a developing chamber that is filled with a small amount of solvent. The solvent cannot be too polar because it will cause spotted compounds on the TLC plate to rise up too fast, while a very non-polar solvent will not allow the spots to move. The polarity of the spots also determines how far it moves on the plate; non-polar spots are higher than polar ones. After spots on the TLC form, the Rf values are calculated and used to analyze the similarity of the compounds.
1.1 Abstract The purpose of quantitative analysis of protein using a spectrophotometer is to measure the concentration of proteins in a given sample. The experiment is conducted by laboratory method (Biuret Test) and using spectrophotometer to analyze the absorbance of reactants at 540 nm, hence determining the concentration of the proteins in a given sample. The purpose of stopped enzyme assay to study B-galactosidase is to determine the effect of temperature and concentrations of substrate on enzyme activity. B-galactosidase breaks down the disaccharide lactose into simple sugars glucose and galactose. However, glucose is a colorless compound hence it has to be substituted with a compound that is detectable by a visible color change.
Each of the analyte will have its own Rf value under certain circumstances. The separation of the phospholipid classes can be improved by two-dimensional chromatography. This technique requires developing the TLC plate in a direction, then dried, and developed in a solvent mixture at a 90 ° the first development (Singh and Jiang,
The mix components through pipetting the reaction mixture should be invert up and down slowly so that the mixture components are equalised. It can also follow by quick spinning in a micro centrifuge to ensure the components in the microtube are mixed equally. Enzymes should be keep in the ice when it is not store in the freezer because heat from the surrounding can cause denaturation of the enzymes and eventually cause them
SAMPLE REQUIREMENT: Type of specimen: Serum (Blood collected in Plain tube and centrifuged to separate serum). PRINCIPLE OF TEST: In the presence of a strong base such as NaOH, picrate reacts with creatinine to form a red chromophore. The rate of increasing absorbance at 510nm due to the formation of this chromophore is directly proportional to the creatinine concentration in the sample and is measured using a Bichromatic (510,600nm) rate technique. To correct for non-specific reaction caused by serum/plasma pseudo-creatinine chromogens, including proteins and ketones, the results for serum or plasma are corrected by -18 μmol/L (-0.2 mg/dL). (2, 4, 14-18) Alkaline pH (NaOH) Creatinine + picrate red chromophore (absorbs at 510nm) EQUIPMENTS & APPARATUS: Siemens Dimension® clinical chemistry analyzer (X pand and RxL Max) Flex reagent cartridge, Assay Cups, tips, pipettes.
The second reason is that the nonmetals have smaller atomic sizes making it easy to attract electrons but difficult to pull them away. 12. A lot of energy is required to break a strong intermolecular bond. This is because atoms in certain compounds have very strong bonds that require energy to break. This explains why some compounds have higher boiling points than others.
Although the astaxanthin content n Haematococcus accounts to more than 2% on dry weight basis, the tough cell wail of cyst cells hinders solvent extraction and intestinal absorption of the pigment from the intact algal biomass. Methods of cell wall disruption which have been applied include mechanical breakage, chemical hydrolysis and lytic enzymes ( Okabue, RN and -ewis MJ 1983 Biotechnol.Lett 5: 731-736., Grung M, D'Souza, FML, Borowitzka M, and Jaaen Jensen S 1992, J appi Phycol 4: 165-171) either denature astaxanthin or are cumbersome and difficult to apply on a large scale. Therefore the present invention provides an alternate improved process for the extraction of carotenoids from encysted Haematococcus cells which facilitated extraction of carotenoids with out homogenisation of cells or use of lytic enzymes. Procedures currently reported for the extraction of astaxanthin from microorganisms are the
Collecting small fractions is essential in column chromatography because they can be combined together; large fractions can lead to multiple compounds in a specific fraction. The purpose of this experiment was to isolate the three components of Excedrin using column chromatography. Thin layer chromatography (TLC) was used to determine when each of the components had been fully eluted from the column. If there was an overlap in fractions between two components, liquid- liquid extraction was done to separate them. The compounds were characterized via NMR instrumentation and the percent recovery for each compound was calculated to determine whether the isolation was
After receiving an unknown mixture, the sample was streaked for isolation onto TSA, blood agar, and MacConkey plates. Each plate serves as a first step to identify the unknowns. The TSA (tryptic soy agar) can be used to do a gram stain, which differentiates gram-negatives from gram-positives, based on the structural make up of the cell wall (Carson, 2015). The blood agar plate is used to test for hemolytic activity, which is useful for distinguishing gram-positives. A MacConkey plate is selective by inhibiting the growth of gram-positives and differential due to the fermentation of lactose by certain gram-negative species.
A Gly-chloride ion boundary is formed since glycine moves slower than chloride ion. However, glycine still runs slightly faster than other proteins. As a result, the glycine keeps pushing the protein towards the chloride ion. In other words, the proteins are trapped between glycine and chloride ion. The proteins form a very tight band inside the stacking gel.