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What are the two most common methods to determine protein concentration?
UV absorbance at 280nm, Chromogenic assays
What are the four various kinds of chromogenic assays?
Biuret method, Lowry method, BCA method, Bradford method
Describe the UV absorbance method at 280nm
Estimate the protein concentration by using a quartz cuvette, Protein solutions will show strong absorption in the 280nm and 210nm regions, 210nm: peptide bonds in the protein, 280nm: Trp, Tyr, and Phe residues in the protein, Uric acid and bilirubin absorb near 280nm and will interfere with the assay
Describe the Biuret assay
Biuret reagent is a light blue solution with Cu²⁺ ions in an alkaline solution, Reagent is mixed with a solution to determine protein presence, If proteins are present, copper ions react with peptide bonds to form a chelate complex, Biuret turns purple (absorbed at 540nm), indicating protein presence
Describe the Lowry assay
Combination of Biuret reaction and Folin-Ciocalteu reaction, Phosphomolybdic tungstic acid is reduced to hetero poly-molybdenum blue by copper-catalyzed oxidation of aromatic amino acids in the peptide under alkaline conditions, Assay is performed at pH 10-10.5
Describe the BCA (bicinchoninic acid) assay
Similar to Lowry assay but uses bicinchoninic acid instead of Folin-Ciocalteu reagent, Mix reagents, add sample, incubate, and read at 560nm, After Cu²⁺ ions are reduced, two BCA molecules chelate with each Cu⁺ ion, forming an intense purple color absorbed at 560nm
Describe the Bradford assay
Mix protein solution with a blue dye that binds to R groups of arginine and aromatic amino acids (Tyr, Phe, Trp), Dye turns red in acidic conditions and is absorbed at 465nm, Amount of blue is proportional to protein concentration from 1.0µg/mL to 1.5mg/mL
What are the advantages and disadvantages of UV absorbance?
Advantages: no sample preparation, non-destructive, good linear range, Disadvantages: accuracy issues due to nucleic acid interference, low sensitivity
What are the advantages and disadvantages of the Lowry assay?
Advantages: high sensitivity, less protein-to-protein variation, Disadvantages: time-consuming, multi-step process, not compatible with detergents, interference from carbohydrates or reducing agents
What are the advantages and disadvantages of the BCA assay?
Advantages: high sensitivity, faster reaction kinetics, Disadvantages: sample must be read within 10 minutes, not compatible with reducing agents
What are the advantages and disadvantages of the Bradford assay?
Advantages: simple and fast, Disadvantages: protein precipitates over time, high protein-to-protein variability, not compatible with detergents
What method can be used to verify if purified proteins are pure?
Protein gel electrophoresis using SDS-PAGE or isoelectric focusing gel, Two-dimensional gel electrophoresis
Define SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Treating proteins with sodium dodecyl sulfate (a detergent) to give them a negative charge, then separating them by size
Define isoelectric focusing gel
Separating proteins based on charge in a capillary tube gel with a pH gradient, Proteins band at their isoelectric point (pH where they have no net charge)
How does an SDS-PAGE gel work?
Add negatively charged SDS molecules to protein, Load samples into SDS-PAGE gel, SDS binds to amino acids, giving a uniform negative charge while heating linearizes proteins, Apply current to push proteins from negative to positive down the gel, Use a control sample as a molecular weight marker, Stain to visualize, Protein bands separate by size
How does isoelectric focusing gel electrophoresis work?
Incorporate an ampholyte solution into a gel, Establish a stable pH gradient in the gel using an electric field, Add protein solution and reapply the electric field, Stain the gel to show proteins distributed along the pH gradient according to their pI values
How does two-dimensional gel electrophoresis work?
Perform isoelectric focusing to separate proteins by pI values, Apply the first gel on top of an SDS-PAGE gel, Separate proteins by size
What are applications of two-dimensional electrophoresis (proteomics)?
Identifying proteins using two-dimensional electrophoresis, Studying the effects of treatment by comparing control and treated cell populations with fluorescent labeling, Mixing and running samples on the same gel to directly compare differences, Scanning the gel at different wavelengths to generate separate images, Comparing intensity and position of spots to determine upregulation, downregulation, or post-translational modifications, Spot excision proteolytic digestion by cutting out proteins of interest for mass spectrometry analysis
What methods can be used to identify a specific protein in a mixture?
Antibody-based identification (Western blotting), Immunoprecipitation assay, Enzyme-linked immunosorbent assay (ELISA)
Define and describe Western blot
Definition: technique used to detect and quantify specific proteins in a sample, known for specificity and sensitivity in identifying proteins based on size and antibody binding, Principle: separates proteins by size, transfers them to a membrane, and detects them using antibody-based labeling, Procedure: Load and separate protein samples on SDS-PAGE under an electric field, Transfer fractionated proteins onto a PVDF membrane for antibody detection, Block the membrane with a neutral protein like BSA or milk casein to prevent non-specific antibody binding, Incubate with a primary antibody specific to the target protein, Incubate with an HRP-labeled secondary antibody that binds to the primary antibody, Add a chemiluminescent HRP substrate and expose to film, The intensity of the band corresponds to the protein's molecular weight and is quantified to determine the protein amount, Results: Western blotting can be performed on a 2D gel with a 2D PAGE or with specific antibodies
Define and describe immunoprecipitation assay
Definition: technique used to detect proteins in their native form and identify associated proteins, Principle: antibody binds to the target protein, forming an antibody-protein complex that is precipitated out of solution using beads, Procedure: Lyse cells or tissues to release proteins into solution, Add a primary antibody specific to the target protein, Add Protein A/G or magnetic beads that bind to the antibody's Fc region, Centrifuge or apply a magnetic field to separate antibody-protein complexes, Wash beads to remove non-specific proteins, Release the target protein for downstream analysis via Western blot, mass spectrometry, or enzyme assays
Define and describe ELISA
Definition: laboratory technique designed to detect and quantify specific proteins, antibodies, antigens, or hormones, known for sensitivity, specificity, and versatility, Principle: antigen-antibody binding with an enzyme-linked detection system, producing a signal proportional to the target molecule concentration, Procedure: Attach a capture antibody or antigen to a 96-well microplate, Add a blocking buffer to prevent non-specific binding, Add a sample containing the target antigen or antibody and incubate for binding, Introduce an enzyme-linked secondary antibody, Add a chromogenic or chemiluminescent substrate that produces a detectable signal, Measure signal intensity using a spectrophotometer or plate reader, Compare results to a standard curve for quantification
What are the limitations of Western blotting?
Antibodies may not recognize proteins in their denatured form, leading to false negatives
What biomarker can be used to study the pharmacokinetics of a therapeutic protein?
Blood sampling at different time points to determine therapeutic protein concentration, Blood concentration analysis using ELISA, chromatography, isotope/fluorescence labeling, or activity-based assays
How can you find a specific protein in a tissue?
Western blot to determine protein tissue distribution, Immunohistochemistry to detect and visualize specific proteins in tissue samples using antibody-based staining
How can protein structure be determined?
Protein sequencing via Edman degradation or mass spectrometry, Secondary structure analysis using spectra between 260-180nm for α-helices and β-sheets, Tertiary and quaternary structure determination via X-ray crystallography, NMR, cryogenic electron microscopy, or computational modeling
How can we assess the side effects and toxicity of protein drugs?
Symptomatic effects: headache, vomiting, diarrhea, dizziness, fatigue, loss of sleep, appetite changes, and depression, Physiological effects: heart rate and blood pressure changes, respiratory issues, and immune suppression, Blood chemistry analysis: protein, glucose, lipid, ion levels, and tissue-specific markers, Urine analysis for kidney function assessment, Long-term impacts on growth, functional deterioration, pregnancy, and child development
