Protein Structure Types and Protein sequencing methods. Specific methods which can detect amount of a single protein

1. PROTEIN SEQUENCING
METHODS
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Presented by: Sepideh Saroughi

2. Four Protein Structure Types
1. Primary Structure
2. Secondary Structure
3. Tertiary Structure
4. Quaternary Structure
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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
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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
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5. Two major direct methods of protein
sequencing
Mass spectrometry
Edman degradation
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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
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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
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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
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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
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11. c. Displacement chromatography
d. Reversed-phase chromatography (RPC)
 Silica
 Surface-modified
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12. e. Size-exclusion chromatography
 Gel filtration chromatography
 Low resolution
 Tertiary & quaternary structure
 Proteins or polymers
 Polysaccharides
f. Ion-exchange chromatography
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13. g. Bioaffinity chromatography
h. Aqueous normal-phase chromatography
 Between reversed-phase chromatography (RP) and organic
normal phase chromatography (ONP)
 Hydrophilic compounds
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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)
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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.
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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
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18. 1.Individual protein immunoprecipitation (IP)
2. Protein complex immunoprecipitation (Co-IP)
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19. 3. Chromatin immunoprecipitation (ChIP)
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20. 4. RNP immunoprecipitation (RIP)
5. Tagged proteins
 Green Fluorescent Protein (GFP) tag
 Glutathione-S-transferase (GST) tag
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21. 3. Western blot
 Protein immunoblot
 Specific proteins in a sample
of tissue homogenate or extract
 Antibody
 Ammunofluorescence
 Buffer = SDS
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23. 4. Protein immunostaining
 Tissue
 Fluorescent
 Peroxidase & Alkaline phosphatase
 Light microscopy
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24. Edman degradation
 Very important reaction
 50 amino acids long
 Cyanogen bromide & pepsin & Trypsin
 98%
 PPE
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25. single-cell western blotting (scWB)
 Measure cell-to-cell variation in protein expression levels
and protein state
 5 main stages
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27. THANKS FOR YOUR
ATTENTION
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