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Biochemistry Assignment Help
The document provides an overview of various methods in protein biochemistry, including protein purification techniques, cell fractionation, chromatography methods, and protein characterization through assays like gel electrophoresis and western blotting. It discusses specific approaches for sequencing proteins and the use of antibodies in detecting and isolating proteins. The document highlights the importance of exploiting biochemical properties for effective protein analysis and purification.
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Biochemistry Assignment Help
- 1. BIOCHEMISTRY ASSIGNMENT HELP Methods in Protein Biochemistry BY GLOBALWEBTUTORS.COM GLOBALWEBTUTORS.COM
- 2. GLOBALWEBTUTORS.COM 5.1 TheArt and Science of Protein Purification 5.2 Working with Oligopeptides: Sequencing and Synthesis 5.3 Protein Structure Determination 5.4 Protein-Specific Antibodies Are Versatile Biochemical Reagents GLOBALWEBTUTORS.COM
- 3. PROTEOMICS BY GLOBALWEBTUTORS High throughput protein biochemical methods to study the proteome Provides insight into how changes in an organism’s physiology alters the proteome GLOBALWEBTUTORS.COM
- 4. Protein Purification Generallyuses freshly isolated cells or tissues Relies on exploiting the inherent qualitative and quantitative differences in biochemical properties of individual proteins Uses specific biochemical assays to uniquely identify proteins of interest among all other proteins contained within a sample to be analyzed Generally detect a product of a chemical reaction performed by the protein of interest GLOBALWEBTUTORS.COM
- 5. Luciferase: An EarlyBiochemical Assay GLOBALWEBTUTORS.COM An enzyme found in bioluminescent organisms Uses luciferin as a substrate GLOBALWEBTUTORS.COM
- 6. CELL FRACTIONATION BYGLOBALWEBTUTORS.COM Cells are broken open and cell extracts (homogenates) are produced. Protein activity is retained. Cell suspensions are prepared by mincing tissue mechanically or enzymatically to generate membrane- bound cells that can be homogenized in one of three ways: Sonication Shearing (with French press) Incubation with mild detergents Centrifugation is performed to increase the concentration and purity of the target protein in the sample. GLOBALWEBTUTORS.COM
- 7. Cell Fractionation: Fraction Formation Fractions are created. Each fraction contains less total protein than the beginning of the cell extract which enriches the target protein. This enrichment can be expressed as specific activity Specific activity = the total amount or activity of the target protein divided by the total amount of protein in the fraction. GLOBALWEBTUTORS.COM
- 8. Post Centrifugation: SaltingOut Used to exploit differences in solubility of the target protein relative to other proteins in the fraction Involves adding increasing amounts of saturated salt solutions to the protein sample This causes the formation of insoluble protein aggregates that are functional when resolubilized. GLOBALWEBTUTORS.COM
- 9. Dialysis Used toremove ammonium sulfate from the protein sample Uses diffusion to leave protein in the buffer the proper ionic strength and pH GLOBALWEBTUTORS.COM
- 10. Chromatography Techniques Column Gel filtration Affinity GLOBALWEBTUTORS.COM
- 11. COLUMN CHROMATOGRAPHY Usedto separate proteins based on different physical or chemical interactions between the proteins and the column matrix Uses a column to separate individual proteins from a sample Separated proteins (fractions) can be collected in small containers GLOBALWEBTUTORS.COM
- 12. Gel Filtration Chromatography Separates proteins based on size Also called size-exclusion chromatography Large proteins migrate faster than smaller proteins, which may get trapped in the carbohydrate beads. GLOBALWEBTUTORS.COM
- 13. High Performance Liquid Chromatography(HPLC) High-resolution version of gravity-based gel filtration chromatography Column matrix contains smaller particle beads and leads to greater separation of proteins that are a similar size. High pressure is required to force buffer and protein through the column. GLOBALWEBTUTORS.COM
- 14. Affinity Chromatography Exploitsspecific binding properties of the target protein to separate it from other cellular proteins that lack this function High-affinity ligand for the target protein is covalently linked to the matrix bead. Other proteins pass through the column. An antibody column can also be used to isolate antigenic proteins. GLOBALWEBTUTORS.COM BEST ASSIGNMENT HELP GLOBALWEBTUTORS.COM
- 15. Gel Electrophoresis Usedto approximate the molecular mass of a protein or if the purified protein includes more than one polypeptide chain Polyacrylamide gel electrophoresis (PAGE) Separates proteins on the basis of charge and size Percentage of gel is important depending on protein size. GLOBALWEBTUTORS.COM
- 16. Edman Degradation, Part1 Improved Sanger’s protein sequencing method Phenylisothiocynate (PITC) is covalently attached to the N-terminal amino acid and then treated with TFA. The polypeptide is cleaved between the first and second amino acid. This is an automated process that can sequence an oligopeptide up to 50 amino acid residues. GLOBALWEBTUTORS.COM
- 17. Protein Cleavage, Part1 For proteins longer than 50 residues, enzymatic cleavage with trypsin and chymotrypsin is performed. Trypsin Cleaves on the C-side of Lys and Arg Chymotrypsin Cleaves at C-side of Tyr, Trp, and Phe GLOBALWEBTUTORS.COM
- 18. Western Blotting Usedto detect proteins separated by gel electrophoresis Uses two antibodies: Primary (protein-specific) Secondary (detection antibody) GLOBALWEBTUTORS.COM
- 19. IMMUNOFLUORESCENCE BY GLOBALWEBTUTORS An antibody-based technique used to identify proteins in cells that have been chemically treated in a way that preserves cell architecture After washing, cells can be visualized directly through fluorescence microscopy if the primary antibody is fluorescent or the secondary antibody contains a fluorescent group. GLOBALWEBTUTORS.COM
- 20. Immunoprecipitation Variation ofaffinity purification Monoclonal antibody is covalently linked to a carbohydrate bead. Co-immunoprecipitation can be performed as well. GLOBALWEBTUTORS.COM
Editor's Notes
- #4 Review the proteome (collection of proteins in an organism).
- #6 Figure 5.1 The firefly enzyme luciferase converts d-luciferin to oxyluciferin in an ATP-dependent reaction that produces light. b. Molecular structure of the luciferase enzyme from the Japanese firefly Luciola cruciata, showing the location of a luciferin analog in the enzyme active site.
- #7 Be sure to reinforce that fresh cells are used. Please explain centrifugation and the importance of balancing the centrifuge.
- #9 State that ammonium sulfate is generally used for salting out.
- #10 Figure 5.6 Dialysis uses a semipermeable membrane to allow equilibration of small molecules across the membrane. By equilibrating against fresh dialysis buffer in the beaker several times, the ammonium sulfate salt is effectively diluted from inside the dialysis bag and replaced with an appropriate buffer. The trapped proteins can then be removed from the dialysis bag and further purified.
- #12 Mention that the column can be plastic or glass cylinder.
- #13 Figure 5.9 Gel filtration chromatography separates proteins on the basis of size. Large proteins cannot enter the pores of the gel matrix beads and elute from the column first. The smaller proteins elute later because they are slowed down by entering the beads.
- #15 State that this only works if the ligand can covalently bind to the matrix bead.
- #16 Be sure to state that high percentage gels resolve small proteins very well, and vice versa. Be sure to also state that PAGE without SDS or other denaturants is called native PAGE.
- #21 Figure 5.39 Antibodies can be used to identify proteins that are associated with protein antigens in large cellular complexes by combining immunoprecipitation with mass spectrometry. Immunoprecipitation uses antibodies that are covalently linked to carbohydrate beads and then applied in a co-immunoprecipitation protocol that enriches for a particular protein and any other proteins with which it is associated. Conventional SDS-PAGE and mass spectrometry are then used to identify all of the proteins in the complex. In this example, each protein is excised separately and analyzed by mass spectrometry as shown. Please explain co-immunoprecipitation.