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 p...

# 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 - UV Spectroscopy-A205 - Fluorescence emission - Amino acid analysis - HPLC - Colorimetric assays - Lowry - Bradford - BCA ## Spectroscopic procedures - **The ELECTROMAGNETIC SPECTRUM** - Penetrates Earth Atmosphere - Wavelength in meters - About the size of - Frequency in Hertz - Temperature of bodies emitting the wavelength in Kelvin - **Spectroscopy** deals with the production, measurement, and interpretation of spectra arising from the interaction of electromagnetic radiation with matter. - **Spectroscopy is the study of the interaction between material and electromagnetic radiation:** - **UV Spectroscopy-A280** - Absorbance of aromatic amino acids - Very purified protein, 20-tug/ml - **Fluorescence emission** - Fluorescence emission of aromatic amino acids - 5-50ug/ml - **UV Spectroscopy-A205** - Absorbance of peptide pond - 1-100ug/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 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 - Collimatot (lens) - Wavelength Selector (slit) - Detector (Photocell) - Light Source - Monochromator (Prism or Grating) - Sample Solution (in Cuvette) - Optical Density: The ability of a laboratory specimen to absorb or block the passage of light - Digital Display or Meter ## How to Use Spectrophotometer? 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. ## Thank you