1. Done by : Rayan Osman Submitted to Dr. Roland Bou Raad 1 Test For Protein Quantification

2. Plan 2 Definition of proteins Ultraviolet-visible spectroscopy Biuret Method The Lowry Method Bicinchoninic Acid Assay (BCA) Dye-Binding Assays 1. Bradford Assay 3. What is Protein? 3 Proteins are complex, specialized molecules composed of carbon, oxygen, hydrogen, nitrogen and sometimes sulfur. The building blocks of proteins are amino acids. There are 20 different amino acids that combine to form polypeptides (proteins). The different amino acids have different side chain, but are otherwise identical. Proteins have many important roles in organisms. Structural proteins such as collagen or elastin, provide support. Regulatory proteins such as enzymes control cell processes. Proteins also play an important part in the immune system (antibodies), oxygen transport (hemoglobin), movement (muscles) etc. 4. Ultraviolet-visible spectroscopy 4 o A quick, but less specific measure of protein concentration is the absorbance of the sample at 280 nm. Most proteins have an absorbance maxima at 280 nm as opposed to nucleic acids which absorb at 260 nm. Aromatic residues, such as tryptophan, tyrosine and phenylalanine are responsible for the observed absorbance, thus proteins lacking these residues cannot be measured using this assay. Requires approximately 50 g for accurate readings. o Although nucleic acid contamination can interfere with measuring protein concentration at 280 nm, a more accurate measure can be taken by measuring the absorbance of the sample at both 260 nm and 280 nm and using the following calculation: o Protein mg/mL = 1.55 A280 0.76 A260 5. Advantages: 5 Good for samples containing a single protein of known molar absorptivity Useful in chromatography to determine elution profile of protein Easy, quick assay Moderate sensitivity 6. Disadvantages: 6 Need spectrophotometer capable of reading in the UV region Need to use quartz Cuvettes Cannot use with proteins that do not contain aromatic residues Nucleic acid contamination can be a problem 7. Biuret Test 7 A test for detecting the presence of peptide bonds. Under alkaline conditions, Cu2+ forms a violet-colored complex. Protein concentration is directly proportional to the intensity of color, absorbed at 540 nm. The Biuret reagent contains sodium hydroxide, hydrated copper (II) sulfate and potassium sodium tartrate (to stabilize the complexes). Reduction of copper results in a color change, which can be read at 550 nm. The linear range is typically 0.5-20mg protein. 8. Concentration range 8 150 to 9000 g/mL (BSA) 9. Biuret Test Negative biuret test positive biuret test (polypeptide chain chelates with a copper ion) 9 10. Advantages: 10 Doesnt rely on the amino acid composition of the protein Does not require a spectrophotometer capable of measuring in the UV region Good for whole tissue samples and other sources of high protein concentration Relatively few materials interfere with the assay Simple procedure. 11. Disadvantages: 11 Buffers, such as Tris and ammonia, can interfere with the reaction Cannot measure the concentration of proteins precipitated using ammonium sulfate Low sensitivity requires higher amounts of protein Nucleic acid contamination can be a problem Proteins with abnormally high or low percentage of aromatic amino acids will give high or low readings. 12. The Lowry Method 12 The development of the Lowry method (named after Oliver Lowry) introduced a more sensitive assay for determining protein concentration. The Lowry method is sensitive, highly reproducible, inexpensive and easy to perform. A modification of the Biuret test, the Lowry method relies on the reaction of copper with proteins, but the sample is also incubated with the Folin-Ciocalteu reagent. Reduction of the Folin-Ciocalteu reagent under alkaline conditions results in an intense blue color that absorbs at 750 nm. The Lowry method is best used with protein concentrations of 0.011.0 mg/mL. Tyrosine, tryptophan, and cysteine of proteins reduce molybdenum acid and phosphotungstic acid of a phenol reagent, turning the solution blue. 13. Concentration range 13 5 to 200 g/mL (BSA) 14. 14 15. 15 16. Advantages: 16 Easy to use Highly reproducible Inexpensive High Sensitivity and broad linear range 17. Disadvantages: 17 Timing and mixing of reagents must be precise Sensitivity depends on composition of protein as reaction partly dependent on polar amino acids Interference from some buffers, particularly detergents Complicated procedure with a long preparation. 18. Bicinchoninic Acid Assay (BCA) 18 BCA is similar to Lowry, except Bicinchoninic acid (BCA) is used instead of the Folin-Ciocalteu reagent. After reduction of Cu2+ ions, two molecules of BCA chelates with each Cu+ ion resulting in formation of an intense purple color that absorbs at 560 nm. BCA is as sensitive as the Lowry method and works well with protein concentrations from 0.5 g/mL to 1.5 mg/mL. Although detergents do not interfere as strongly as in the Lowry method, other contaminants can interfere with the reaction. In addition, aromatic amino acids can influence the reaction. However, at higher temperatures (37-60C), peptide bonds can also contribute to formation of the product. Therefore it is recommended to perform the reaction at higher temperatures to increase the sensitivity and decrease the variability due to amino acid composition. 19. Concentration range 19 20 to 2000 g/mL (BSA) 20. 20 21. 21 22. Advantages: 22 Easy to use Highly reproducible Inexpensive High sensitivity Simple procedure. 23. Disadvantages: 23 Interference from carbohydrates, catecholamines, tryptophan, lipids, phenol red, cysteine, tyrosine, impure sucrose or glycerol, uric acid, iron and hydrogen peroxide Color continues to develop over time, but is stable for measurement after 30 minutes at 37C 24. Dye-Binding Assays 24 Dye binding assays rely on an absorbance shift that occurs when a dye binds to proteins. Several different dyes can be used: Coomassie Brillant Blue G-250, bromocresol green, pyrogallol red. The most commonly used dye-binding assay is the Bradford Assay. 25. Bradford Assay 25 The Bradford assay, is an easy, sensitive and accurate method for protein quantification. Binding of Coomassie Brillant Blue G-250 to proteins, causes a shift of the dye from red (465 nm) to blue (595 nm) under acidic conditions. It is compatible with more common reagents, although detergents can cause interference. Proteins with a concentration of 20-2000 g/mL can be measured using the Bradford assay. 26. Concentration range 26 10 to 2000 g/mL (BSA) 27. 27 28. 28 29. Advantages: 29 Easy to use Sensitive Quick Compatible with many buffers Very simple operation. 30. Disadvantages: 30 Reagent stains cuvettes Often need to dilute samples prior to analysis Depends strongly on amino acid composition Sensitive to detergents although some companies offer detergent-compatible Bradford reagents 31. WST 31 Reduce WST-8 with proteins with a high pH, turning the sample blue. Uses the absorption maximum at 650 nm to determine the quantity 32. Concentration range 32 50 to 5000 g/mL (BSA) 33. 33 34. 34 35. Advantages Simple operation. Hardly influenced by a surfactant. 36. Disadvantages The chromogenic rate differs for each protein.