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Biochemistry Lab (Quiz #2)

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Three important reasons to know concentration of protein in a sample:
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1. Characterization of proteins and enzymes 2. physiological studies of protein expression 3. clinical diagnosis of altered protein levels in body fluids (indicate disease).
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Progress of purification can be tracked by..
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Comparing total amount of desired protein antigen (or other specific method of the target protein), and the total amount of protein after each fraction step.
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If purification is proceeding properly..
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The amount of enzymatic activity to the total protein concentration increases.
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Specific activity
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Ratio of enzymatic activity to the total amount of protein in a sample (measured in units per mg of protein).
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Two methods are required for characterization of proteins measurement of specific activity:
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1. A method for determination of total protein 2. A method for determination of the units of activity.
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Factors to consider when choosing an assay
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1. Range of useful response 2. Sensitivity and specificity of assay 3. The ease of conducting the assay 4. Safety considerations 5. Cost of assay
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Range of useful response
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Refers to whether the change in response to changes in a sample can be measured at various concentrations of material.
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Linear response
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Applies over a very broad range of protein concentrations. This is ideal but not REQUIRED.
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If an assay gives a good response only over a narrow range of protein concentrations..
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You need to dilute the sample.
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Sensitivity
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Refers to the lowest level sample can be detected.
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Specificity
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Refers to the assay only responding to the desired component (protein) and not to others present. The protein solution can be contaminated with other proteins present, lipids, carbohydrates, nucleic acids, and more.
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Compounds known to affect chromogenic reactions:
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Detergents, carbohydrates, nucleic acids, lipids, and salts. (more than that)
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Results with chromogenic assays are only an..
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Estimation when dealing with proteins.
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Non-colorimetric procedures for protein quantification:
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1. Amino acid analysis of proteins using acid hydrolysis followed by separation of amino acids. 2. Determination of nitrogen derived from proteins (Johan Kjeldahl). 3. Determine the dry matter content after drying at constant temperature at constant rate. 5. Growing cells in presence of radioactive amino acids to incorporate into proteins.
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Colorimetric assay advantages:
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1. Availability of spectrophotometers 2. Great accuracy 3. Specificity of colorimetric assays 4. Low cost 5. Simple and safe
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Absorbance at 280 and 260 nm
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Concentration of protein can be estimated by measuring the absorbance of protein-containing solutions at 280 nm. This reading in the UV spectrum is due to aromatic amino acids. Advantages: Does not destroy sample, very rapid. Disadvantages: A spectrophotometer that can read into the UV region is required. Not accurate unless protein is pure and molar absorptivity is known. Very sensitive to nucleic acids due to their maximum absorption being at 260 nm.
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Biuret Method of Protein Determination
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Using alkaline condition , copper (II) binds with peptide nitrogens of proteins and this complex absorbs light maximally at 550 nm. Advantages: Copper reacts with peptide bonds so there are little interferences from free amino acids. Composition of amino acids is not important. Disadvantages: Tris and ammonia react with copper and interfere with assay. The assay is insensitive and only useful at high concentrations.
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Lowry Method of Protein Determination
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Uses Biuret reaction followed by reduction under alkaline conditions of Folin Ciocalteu reagent. Yields and intense blue product with maximum absorbance at 750 nm which is usually out of range of interfering colors. Advantages: Inexpensive, easy to perform, very sensitive, and highly reproducible. Disadvantages: Sensitive to a variety of contaminants. Standard curves are linear at low pH, and timing and mixing of reagents must be precise. Sensitivity to this depends on concentration of protein.
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Bicinchoninic Acid (BCA) Method for protein determination
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Similar to Lowry reaction except BCA is used instead of Folin-Ciocalteu. Results in intense purple color with absorption at 560 nm. Color develops over time, needs to incubate for 30 mins at 37 degrees celsius. Advantages: Disadvantages: Sensitivity is similar to Lowry but not as sensitive to certain contaminants (such as detergent but more sensitive to carbs). H2O2, uric acid, and iron interfer.
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Dye-Binding Method (Bradford Method) of protein determination:
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Coomassie Blue (G-250) binds to proteins and causes a shift in absorbance from Amax of 465 nm (red) to 595 nm (blue) in acidic conditions. Dye is added to samples and A is measured at 595 nm. Advantages: Does not maintain linearity over wide range of protein concentrations (sensitive and accurate). Disadvantages: High concentrations of detergent interfere with assay.
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Other dyes used in dye-binding method:
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Pyrogallol Red, and Bromocresol Green.