Protein sequencing methods
- 1. PROTEIN SEQUENCING METHODS 1 Presented by: Sepideh Saroughi
- 2. Four Protein Structure Types 1. Primary Structure 2. Secondary Structure 3. Tertiary Structure 4. Quaternary Structure 2
- 3. Genetic methods The function of parts of proteins can be better understood by studying the change in phenotype as a result of this change. Inserting protein tags Such as the His-tag Modified protein 3
- 4. Protein extraction from tissues liquid nitrogen Protein purification 1. Protein isolation 2. Protein extraction and solubilization 3. Concentrating protein solutions 4. Gel electrophoresis 5. Electrofocusing 4
- 5. Two major direct methods of protein sequencing Mass spectrometry Edman degradation 5
- 6. Non-specific methods that detect total protein only Absorbance: 100 μg/mL to 1 mg/mL Bradford protein assay: ~1 mg/mL Biuret Test Derived Assays: 1. Bicinchoninic acid assay (BCA assay): 0.5 μg/mL 2. Lowry Protein assay: 0.01–1.0 mg/mL Fluorescamine: Amido black: 1-12 μg/mL Colloidal gold: 20 - 640 ng/mL Nitrogen detection: 1. Kjeldahl method 2. Dumas method 6
- 7. Specific methods which can detect amount of a single protein Spectrometry methods: 1. High-performance liquid chromatography (HPLC) 2. Liquid chromatography–mass spectrometry (LC/MS) Antibody dependent methods: 1. Enzyme-linked immunosorbent assay (ELISA) 2. Protein immunoprecipitation 3. Western blot 4. Protein immunostaining 5. Immunoelectrophoresis 7
- 8. Spectrometry methods 1. High-performance liquid chromatography (HPLC) High-pressure liquid chromatography Separate, identify, and quantify each component in a mixture Manufacturing Legal Research Medical 8
- 9. Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent. These interactions are physical in nature, most often a combination. Types: a. Partition chromatography b. Normal–phase chromatography c. Displacement chromatography d. Reversed-phase chromatography (RPC) e. Size-exclusion chromatography f. Ion-exchange chromatography g. Bioaffinity chromatography h. Aqueous normal-phase chromatography 9
- 10. a. Partition chromatography Paper chromatography b. Normal–phase chromatography Silica Chloroform 10
- 11. c. Displacement chromatography d. Reversed-phase chromatography (RPC) Silica Surface-modified 11
- 12. e. Size-exclusion chromatography Gel filtration chromatography Low resolution Tertiary & quaternary structure Proteins or polymers Polysaccharides f. Ion-exchange chromatography 12
- 13. g. Bioaffinity chromatography h. Aqueous normal-phase chromatography Between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP) Hydrophilic compounds 13
- 14. 2. Liquid chromatography–mass spectrometry (LC/MS) HPLC & MS Biotechnology, environment, food processing and pharmaceutical, agrochemical, and cosmetic industries Electrospray ionization (ESI) Atmospheric pressure chemical ionization (APCI) Atmospheric pressure photo- ionization (APPI) 14
- 15. Antibody dependent methods Antibody (Ab) = Immunoglobulin (Ig) 1. Enzyme-linked immunosorbent assay (ELISA) Solid-phase Liquid sample Performing an ELISA involves at least one antibody with specificity for a particular antigen. 15
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- 17. 2. Protein immunoprecipitation Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure. Direct capture & Indirect capture Types: 1. Individual protein immunoprecipitation (IP) 2. Protein complex immunoprecipitation (Co-IP) 3. Chromatin immunoprecipitation (ChIP) 4. RNP immunoprecipitation (RIP) 5. Tagged proteins 17
- 18. 1.Individual protein immunoprecipitation (IP) 2. Protein complex immunoprecipitation (Co-IP) 18
- 19. 3. Chromatin immunoprecipitation (ChIP) 19
- 20. 4. RNP immunoprecipitation (RIP) 5. Tagged proteins Green Fluorescent Protein (GFP) tag Glutathione-S-transferase (GST) tag 20
- 21. 3. Western blot Protein immunoblot Specific proteins in a sample of tissue homogenate or extract Antibody Ammunofluorescence Buffer = SDS 21
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- 23. 4. Protein immunostaining Tissue Fluorescent Peroxidase & Alkaline phosphatase Light microscopy 23
- 24. Edman degradation Very important reaction 50 amino acids long Cyanogen bromide & pepsin & Trypsin 98% PPE 24
- 25. single-cell western blotting (scWB) Measure cell-to-cell variation in protein expression levels and protein state 5 main stages 25
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