Determination of Total Protein Content

MATERIAL III DETERMINATION OF TOTAL PROTEIN CONTENT (QUANTITATIVE) Basic theory Proteins are macromolecules that make up more than half of cells. Proteins determine the size and structure of cells, are the main components of the communication system between cells and act as catalysts for various biochemical reactions in cells. That's why most biochemical research activities focus on proteins, especially hormones, antibodies and enzymes. All types of protein consist of a series and combination of 20 amino acids. Each type of protein has a unique number and sequence of amino acids. In cells, proteins are found in both the plasma membrane and internal membranes that make up cell organelles such as mitochondria, endoplasmic reticulum, nucleus and Golgi bodies with different functions depending on their location. The proteins involved in biochemical reactions, most of which ,are enzymes, are found in the cytoplasm and some are found in compartments of cell organelles. Proteins are a very heterogeneous group of biomacromolecules. When they are outside living creatures or cells, proteins are very unstable. Each type of protein is characterized by: 1) Distinctive chemical composition Each individual protein is a pure compound 2) Typical molecular weight All molecules in a particular example of pure protein have the same molecular weight. Because the molecules are large, proteins easily undergo physical changes or biological activity. 3) Typical amino acid sequence The amino acid sequence of a particular protein is genetically detailed. However, there are small changes in the amino acid sequence of a particular protein Proteins play an important role in various biological processes. These roles include: 1) Enzymatic catalysis Almost all chemical reactions in biological systems are catalyzed by enzymes and almost all enzymes are proteins. 2) Transport and storage Various small molecules and ions are transported by specific proteins. For example, oxygen transport in erythrocytes by hemoglobin and oxygen transport in muscles by myoglobin. 3) Coordination of movements. Muscle contractions can occur due to the shifting of two protein filaments. Other examples are the movement of chromosomes during mitosis and the movement of sperm by flagella. 4) Mechanical support Skin and bone tension is caused by collagen which is a fibrous protein. 5) Immune protection Antibodies are very specific proteins and can recognize and combine with foreign objects such as viruses, bacteria and cells from other organisms. 6) Generate and transmit nerve impulses. The response of nerve cells to specific stimuli is mediated by receptor proteins. For example, rhodopsin is a light-sensitive protein found in retinal rod cells. Another example is receptor proteins in synapses. 7) Regulation of growth and differentiation In higher organisms, growth and differentiation are regulated by growth factor proteins. For example, nerve growth factor controls the growth of nerve tissue. Additionally, many hormones are proteins Biuret method Photometric test based on the biuret method. Simultaneously copper ions and protein form a blue violet complex in an alkaline solution. The absorbance of this colored complex is directly proportional to the protein concentration in the sample. Procedure 1. Wavelength : Hg 546 nm ( 540 nm ) 2. Spectrophotometer : 500 nm 3. Incubation temperature : 20 – 25°C or 37° Material : 1. Blood Sample 10-15 ml Reactor : 1. Plasma / blood serum 2. Reagent 1 - Sodium hydroxide - Potassium sodium tartrate 3. Reagent 2 - Sodium hydroxide - Potassium sodium tartrate - Potassium iodide - Copper sulfate Specimen (biological sample) Serum, plasma heparin or plasma EDTA Stability: • 6 days at a temperature of 20-250C, • 4 weeks at temperature 4-80C and • <1 year month at temperature -200C Tool : 1. Reaction tube 2. EDTA 3. Centrifuge 4. Kits 5. Spectrophotometer 5.Myropipette Implementation: Preparation of working reagents Pipette in cuvette Black Standard Sample Sample, std - 20 µl 20 µl aquabides 20 µl - - reagent 1 1000 µl 1000 µl 1000 µl Mix, measure the absorbance of A1 against the reagent blank after 1-5 minutes at a temperature of 20-25°C /37°C and add Reagent 2 250 µl 250 µl 250 µl Mix, incubate for 5 minutes at temperature 20-250C /370C. and measure the absorbance of A2 against the reagent blank within 60 minutes. (∆A) Sample Pipette in cuvette Black Standard Sample Sample, std - 20 µl 20 µl aquabides 20 µl - - reagent 1 1000 µl 1000 µl 1000 µl Mix, standards and samples against blank coreagent within 60 minutes. (∆A) incubation 5 minutes at temperature 37°C. Measure absorbance Nb: Make itworking reagentby mixing 4 parts of reagent 1 with 1 part of the reagent