Reviewer For LBYBIOJ MIDTERMS
concentration of the substance and the path length of the light through the solution.
Standard Curve Preparation for Determining Protein Content
Cell Fractionation and Separation
Protein Quantitation — Quantitation — isolation, characterization, purification, and identification
Homogenization, Homogenization, to break open cells to separate their structural and molecular components.
Procedures that need qunatified protein samples: • chromatography • electrophoresis • functional assays • immunochemical separation analyses
Homogenization Methods • Detergents (SDS) • Salts for Osmotic Alteration • Enzymes (trypsin and proteinase K) • Mechanical methods • Ultrasonification (sound waves)
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280 nm, nm, abs of protein molecules in solutions due to presence of aromatic amino acids (tyr & trp) Zero Buffer Absorbance, Absorbance , to resolve interference of substance in the buffer for absorbance.
Fractionation, separation or subcellular organelles by centrifugation (by density) Pellet — material that collects at bottom of microfuge tube Supernatant — fluid above the pellet
• Crude, homogenized material, all Colorimetric Methods of Quantitation • Protein dye-binding Chemistry (Coomassie/Bradford) • BCA™ Protein Assay • Modified Lowry Protein Assay • Protein-copper Chelation Chemistry Selection of Protein Standard • highly purified versi on of predominant protein in sample • Bovine Serum Albumin (BSA)
most
Biuret Assay • Biuret, small compound formed when urea is heated causing two urea molecules to join. Copper complexes produced produce strong blue color. Bradford Assay • Coomassie Brilliant Blue G-250 —binds to basic (especially Arg) and aromatic amino acid residues • Cationic (Red) [470 nm] • Neutral (Green) • Anionic (Blue) [595 nm] Beer’s Law, the quantity of light absorbed by a substance dissolved in a fully transmitting solvent is directly proportional to the
components present • Nuclei (pellet) • Soluble (supernatant) • Microsome (pellet) 595 nm, abs to be read at
Marker Enzyme Assay Marker Enzymes — enzymes localized in a particular organelle of the cell A. Alkaline Alkaline Phosph Phosphodies odiesterase terase (APDE) (APDE) Acid • present in phagocytic vacuoles • thymidine 5’ monoP —> p-nitrophenol • yellow compound
• Buffer D (Tris-borate, Triton X-100, MgCl2, ZnSO4) • Substrate D (p-nitrophenyl thymidine 5’ monophosphate)
B. Peroxidase Assay • present in peroxisomes • Peroxidase oxidized reduced TMB in presence of H2O2 • Blue color
SDS - Sodium Dodecyl Sulfate • gives proteins net negative charge • removes 2ndary and 3rtiary structures Ammonium Persulfate and TEMED “POISONOUS”
• Tetramethyl Benzidine (TMB) C. Acid Phosphatase Assay
• Present in Lysosomes • pNPP or pNTP —> para-nitrophenol • Yellow Compound • Buffer C (glycine-HCl, Triton X-100) • Substrate C (p-nitrophenol phosphate) D. Mit och ond ria l Red uct ase / Dehydrogenase Assay • Reduction of Blue Resazurin —> Red Resofurin
• Alamar Blue® Viability Reagent E. Protease Acitvity Assay
• Protease, may connote some physiological conditions such as stress • Differential Scanning Fluorimetry (DSF) • Proteases digest BSA, products bind to Flamingo™ —> enhanced fluorescence • Relative Fluorescence Units (RFU), measure of fluorescence
• BSA or Chicken Egg White Albumin • Flamingo™ Pink Fluorescent Stain DNAse — key enzymes released during apoptosis, responsible for damaging DNA SYBR Green — dramatic fluorescence in presence of double stranded DNA
SDS-Polyacrylamide Gel Electrophoresis Gel Electrophoresis — separates charged molecules by running through a matrix (gel) in an electrical current. Nucleic Acids — net negative charge due to their phosphate group Cathode (NEGATIVE), Anode (POSITIVE)
Mini-Protean III — vertical slab unit designed for faster electrophoresis
• 4% Acrylamide — Stacking Gel, restricts protein migration, concentrate and properly align protein samples • 12% A crylamide — Running Gel, separates individual polypeptides into discrete bands Staining • Coomassie Blue R250 • SYPRO Ruby
SDS-PAGE Gel Image Analysis Protein Profiling — data from SDS-PAGE used to determine how closely related two or more species are at the level of expressed gene products
Protein Sequence Analysis and Homology Modeling of 3D Structures Reverse Genetics — Once the gene sequence is known, the exact amino acid sequence can be deciphered and thus will show more the inherent properties of the protein being investigated. !mino
Acid = Residue Amino Acid – alpha carbon, carboxylate group, amino group, R group (gives property) Affects Structure of the Protein e.g proline, ‘chain breaker’ because of its cyclic structure glycine, mabilis magfold lol Primary Structure - amino acid sequence Secondary Structure – structure, folding
Types of Secondary Structures 1.alpha helix – glycine on turn position, 3.6/3.4 amino acid residue per turn. Maintained by Hydrogen bonding 2. beta sheath 3. Random coil – randomly coiled, still a type of secondary structure 4. Turn – not necessarily for the alpha helix Tertiary Structure – 3d Quaternary Structure - subunits Dimeric, trimeric, multimeric, etc. Xray Crystallography/Protein Crystallography – determining 3d protein structure **you need to have a protein signal Crystal – ordered periodic material **more reliable than NMR NMR – Nuclear Magnetic Resonance Concept: Poly exclusion principle Spots correspond to the structure of the protein Downside: difficult to interpret *like taking a video of the protein, measuring the structure in solution. Why bother? Structure-function relationship Homology Modelling - Refers to the process wherein the structure of the protein is predicted based on a template - The template must be well-known, a crystal structure (?) - Structures are conserved within the family of the proteins evolution Ramachandran Plot - Plots phi and psi angles, corresponds to the tortional angles of the peptides o Alpha carbon and the nitrogen Tortional angle – between alpha carbon and nitrogen Psi angle – between carbonyl carbon and alpha carbon
Omega Angle – not included in the ramchandran plot Between carbonyl carbon and nitrogen amide bond It does not give you any idea about the protein structure Becausde of its rigidity (partial double bond character), hindi masyadong nakakaikot
