Protein Quantification Lab 8 PDF

# Protein Quantification "Lab. 8" ## Biochemical and Physiological Genetics Course (English Program) Level 2 ## Outlines - To identify following expressions - Introduction to spectrophotometer - How to use spectrophotometer? - Protein Quantification Methods - Spectroscopic pro...

# Protein Quantification "Lab. 8" ## Biochemical and Physiological Genetics Course (English Program) Level 2 ## Outlines - To identify following expressions - Introduction to spectrophotometer - How to use spectrophotometer? - Protein Quantification Methods - Spectroscopic procedures - Amino acid analysis - Colorimetric assays ## Protein Quantification - It is the measurement of protein concentration in an aqueous sample - It is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. ## Protein Quantification Methods ### Spectroscopic Procedures - UV Spectroscopy-A280 - Absorbance of aromatic amino acids - Very purified protein, 20-300ug/ml - UV Spectroscopy-A205 - Absorbance of peptide bond - 1-100ug/ml - Fluorescence emission - Fluorescence emission of aromatic amino acids - 5-50ug/ml ### Amino Acid Analysis (AAA) - It is considered the method of choice to determine the purity and chemical composition of a protein or peptide. - Amino acid analysis (AAA) is a method for breaking down a protein or peptide into its components (amino acids) and determining their identities and relative quantities of the freed amino acids. - Required amount of sample is 0.05 nmol or 2.5 ug - The AAA procedure includes hydrolysis, derivatization, separation, detection and quantification. - PITC: Phenylisothiocyanate - PITC reacts with the free amino groups in amines and amino acids making them suitable for UV-detection through performing RP-HPLC. - RP-HPLC.: Reversed Phase-High Performance Liquid Chromatographic Method ### Colorimetric Assays - Methods of determining the concentration of a chemical element or chemical compound in a solution with the aid of a color reagent. - The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques. #### 1. Lowry Method - Under alkaline conditions the divalent copper ion (Cu+2) forms a complex with peptide bonds in which it is reduced to a monovalent ion (Cu+). - Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungsten blue (650 to 750 nm) #### 2. Bis-Cinchinonic Acid (BCA) - Is based on two chemical reactions: - First, the peptide bonds in protein reduce Cu2+ ions from the copper(II) sulfate to Cu+ (a temperature dependent reaction). - Next, two molecules of bicinchoninic acid chelate with each Cu+ ion, forming a purple-colored complex that strongly absorbs light at a wavelength of 562 nm. #### 3. Bradford Method - Is based on: - An absorbance shift of the dye Coomassie Brilliant Blue G-250. The Coomassie Brilliant Blue G-250 dye exists in three forms: anionic (blue), neutral (green), and cationic (red). - Under acidic conditions, the red form of the dye is converted into its blue form, binding to the protein being assayed. ## Protein Assays Sensitivity - A280 (20 to 3000 ug/ml) - A205 (1 to 10 ug/ml) - Fluorescence (5 to 50 ug/ml) - Lowry method (5 to 100 ug/ml) - Bradford method (10 to 100 ug/ml) - BCA (bicinchoninic acid) method (0.5ug/ml) ## Spectroscopic Procedures - Spectrophotometry is an experimental technique that is used to measure the concentration of solutes in a specific solution by calculating the amount of light absorbed by those solutes. - Spectrophotometer is an apparatus for measuring light intensity in apart of the spectrum, as transmitted or emitted by a particular substance. ## Spectrophotometer - According to available wavelengths, there are several types: - Vis spectrophotometers - UV spectrophotometers - Infrared spectrophotometers - UV-Vis spectrophotometers - UV-Vis near-infrared spectrophotometers (UV VIS NIR) ## Principles of Spectrophotometry - **Optical Density:** The ability of a laboratory specimen to absorb or block the passage of light ## How to Use Spectrophotometer? - Most spectrophotometers need to warm up before they can give an accurate reading then clean the cuvette. - **1.** Turn on the machine and let it sit for at least 15 minutes before running any samples. - **2.** Use deionized water. - **3.** Calibrate the machine with the blank and choose and set the wavelength of light to analyze the sample with. - **4.** Place the test sample inside. - **5.** Wait 10 seconds before recording the values of % transmittance and/or absorbance. - **6.** Measure the absorbance of your experimental sample 3 times and analyze obtained data.