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Lecture 3

BIOLOGY 2B03 Lecture Notes - Lecture 3: Cytosol, Transfer Rna, Cell Membrane


Department
Biology
Course Code
BIOLOGY 2B03
Professor
Rosa Da Silva
Lecture
3

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2B03- Module 3: Biomembranes and Cell Architecture
Purification of Proteins
- Ways to purify protein: Structural analysis, determine the AA sequence, functional analysis or develop antibodies to a
protein
1) Develop a good assay
2) Select a protein source
3) Break open cells- protein extract
4) Solubilize protein
5) Stabilize protein
6) Fractionate
7) Determine purity
8)
9) Protein Assay
10) Protein assay- way to detect presence and [ ] of the protein
- How to detect the protein
oUnique to the protein to avoid confusion
11) Ex. Look at enzymatic activity for an enzyme; Immunological assay to look at presence and [antibody]; look at presence
of protein through biological activity/function if it binds to a specific substrate
12)
13) Protein Source and Extraction
- Easy extracted in large amounts
oTissues, cultured cells, overexpression in another system
- Contain high [protein of interest], and low [proteins that may co-purify] and [proteases]
oOr could add protease inhibitors
- Prefer: express protein in alternative cell type (ex. mouse protein in bacterial cells)
- Then, lyse cell to extract protein
14)
15) Protein Solubilisation
- Cytosolic or secreted proteins are soluble and, transmembrane and membrane-associated proteins aren’t
oHard to extract transmembrane proteins
- Solubility dependent on: pH of solution, [salt] and the presence of detergents
16) Detergents- increase the solubility of insoluble proteins in a solution
17)
18) Protein Stabilization
- Want to stabilize to ensure protein maintains native structure/noncovalent interactions and doesn’t get degraded
19)
20) Parameters:
1. Temperature
2. Protease inhibitors
3. Ligands
4. Salts
5. Metal ions
6. Concentration
7. pH

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8.
9. Protein Fractionation
10. Fractionation- process of separating proteins into different groups/fractions based on attributes
- Attributes: size, polarity, charge, solubility, shape
- Proteins have one of the above unique to them, so multiple fractionation processes are used to distinguish between
many proteins
-SIZE: gel electrophoresis, gel filtration chromatography, ultracentrifugation
-CHARGE: ion exchange chromatography, gel electrophoresis
-POLARITY: adsorption chromatography, hydrophobic interaction
chromatography
-SPECIFICITY OF BINDING: affinity chromatography
11.
12. Differential Centrifugation:
13. Separate based on mass and density
1) Tube of crude cell extract: centrifuge at 1000g
2) Transfer supernatant to fresh tube
3) Centrifuge supernatant at 104g; Repeat
4) Centrifuge supernatant at 105g
14.
15. Separation by Chromatography:
16. Separation based on degree of interaction with the beads
- Column contains matrix or beads
oA lot of interactions: move through column slowly
oLes interactions: move quickly
17.
A. Ion Exchange Chromatography:
18. Separate protein based on their charge
- The beads within the column are +ively charged
o+vely charged proteins move quickly
o–vely charged proteins move slowly
- To extract the –vely charged proteins, it has to be
washed with salt wash solution, warm wash solution or
change pH of wash solution, or have –vely charged
beads inside column
19. Ex. Ras protein: separate via affinity chromatography
using GTP or separate by size
20.
B. Gel Filtration Chromatography
21. Separate protein based on size
- The beads have small holes and cavities
oSmall proteins get trapped and move slowly
oLarge proteins flow quickly through the column
- Small beads can be extracted via wash or spinning at low
speeds
- The beads’ holes are not the same size: beads are defined by
their threshold size ie. the largest protein that could fit
22.
23.
C. Affinity Chromatography
24. Separate proteins based on their specificity of binding to another molecule
- The beads are covalently attached to an antibody with a single
protein, called antigen
- Protein of interest will bind with the antibody and the others will
flow through
- Can extract the protein by changing pH, rising T or increasing
[salt of wash solution
- Can also be done with a target that’s not an antibody
25.
26.
27.
28. SDS-PAGE Electrophoresis:
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29. Sample Preparation
- SDS (sodium dodecylsufate) denatures proteins and coats them to have
a –ve charge => effects shape and charge density
30. Protein Separation
- Protein mixture is loaded onto polyacrylamide gel, and begin moving to
the positive end
- Speed depends on molecular weight
- Different lanes in the gel correspond to different fractionation
oBy the last lane, there is one protein remaining (~40 kD)
31. Western Blot
- After separation, they can be transferred to a membrane containing the
protein of interest’s antibody
- Used to separate proteins in complex mixture
32.
33. Multi-step Purification 34. Specific activity = enzyme activity / amount of protein (mg)
35.
1) Precipitation: precipitate some proteins out of extract
a. Total activity decreased because of accidental loss of protein; specific activity increased because of intentional
loss of unnecessary proteins
b. Volume decreased
2) Ion- exchange chromatography
a. Volume decreased
b. Total activity decreased by specific activity increased
3) Size exclusion chromatography and Affinity chromatography
a. Volume decreased
b. Decrease in total activity and increase in specific activity
36. Result: high final specific activity because only protein of interest remains
37. Visualizing the Structure of the Cell
- Limits of the human eye: 1/5 mm = can’t observe objects that close or that small
- Light microscope: uses visible lights; conventional and fluorescent
- Electron microscope: uses beans of electrons to see objects < 100 nm; transmission and scanning
38.
39. Limit of Resolution, D
40. D- the minimum distance between two objects that can be distinguished from one
another
- The smaller the D, the better resolution
41. Wavelength of light: shorter wavelengths of light give higher resolution
42. Numerical aperture, NA: measure of ability to gather light and resolve specimen detail
- Ideal to minimize refraction of illuminating light
43.
44. Improving Resolution: Decrease D
45.
↓ λ
D better resolution
46. Light Microscopy:
- Visible light: 400nm – 700 nm
47. Ex. D = 0.61
λ
/ NA = 0.61(45) / 0.94 = 292 nm; if the objects were closer than 292nm, they would be
indistinguishable
48. Limit of resolution is 292nm.
- Bight field microscopy has resolving power of 0.2um and magnify 1000x
- Samples can be live vs. fixed, or stained vs. unstained
49. UV Light:
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