Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

CHEM 441A+B > Chapter 5: Methods in Protein Biochemistry > Flashcards

Chapter 5: Methods in Protein Biochemistry Flashcards

1
Q

What is the Proteome?

A

The entire collection of proteins found in an organism

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

How is Cell Fracturation prepared?

A
  1. Cells are broken which produces cell extracts (homogenates)
  2. Cell suspension prepared by mincing via 1/3 ways
    a. Sonication - disrupts cell membranes through vibrational ultrasonic waves
    b. Shearing - forcing cells through small opening
    c. Incubation with mild detergents - disrupts cell membrane
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Once the Cell Fracture has been prepared how is centrifugation carried out?

A
  1. Once target protein is isolated it undergoes preparative centrefugation
  2. Four fractions containing Nuclei, mitochondria, plasma membrane, and cytosol
  3. An estimate for the total amount of protein can be made by measuring sample absorbance at 280 nm
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Why is absorbance measured at 280 nm?

A

Because it is the wavelength where tryptophan most absorb light

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is salting out?

A

A process to separate proteins
- Add increasing amounts of saturated salt solution to form ionic interactions with water
- Results in insoluble aggregates of proteins
- Often use (NH4)2SO4

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is dialysis?

A
  • A process of removing a salt (ammonium sulfate) from the protein sample
  • small pores in tubing which allow salt to diffuse out but protein stays trapped
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is Column Chromatography?

A

A process which separates proteins based on chemical or physical interactions
- A column with a matrix in combination witha continuous flow of buffer causes separation of protein
- Measure protein by 280 nm
- Fractions are then collected in small containers

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is Gel Filtration Chromatography?

A
  • Also called size exclusive chrom.
  • separates proteins by size
  • Beads have pores which means there is selectivity for which proteins travel
  • large proteins travel through while small proteins get stuck(delayed) in carbohydrate beads
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How does protein size factor into elution profile?

A

The larger the protein the faster it elutes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is HPLC?

A
  • High Performance Liquid Chromatography
  • Column contains very small beads leading to better separation
  • High pressure needed to force buffer and protein through column
  • Expensive
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is Ion-Exchange chromatography?

A
  • Uses difference in charges of proteins
  • Can have anion or cation charged matrix
  • Charged beads will allow same charged proteins to travel freely while oppositely charged proteins are bound until elution buffer is run.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is Affinity Chromatography?

A
  • ## Uses the binding properties of a target protein to separate it from proteins that lack the same properties
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is Ni-Affinity Chromatography?

A
  • Protein starts or ends with 6 His
  • Binds to Ni
  • Elute with Imidazole buffer
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Rank the efficiency of purification of Chemical methods

A
  • Preparation of a crude cell extract
  • Subcellular fraction using centrifugation
  • Ammonium sulfate precipitate
  • Gel-filtrate Chromatography
  • Ion-Exchange Chromatography
  • Affinity Chromatography
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is Gel Electrophoresis?

A
  • Cheap
  • Apparatus is used to determine protein purity
  • provides an approximate mass of proteins (subunit composition)
  • PAGE (Polyacrylamide gel electrophoresis)
    • PAGE = separates proteins based on charge
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Describe the setup of a PAGE electrophoresis and what it does.

A
  • Electric field pulls proteins according to charge
  • Gel matrix hinders proteins based on size and shape
  • Velocity = (E*z)/f (f = 6pi)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What is SDS-Page

A
  • SDS is a detergent that adds net negative charge to each protein to aid in the migration toward the anode
  • Provides uniform charge
  • Denatures shape so only size is accounted for
  • Small molecules move the fastest
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What process is done to make the protein bands visible?

A
  • Staining with coomassie Brilliant Blue G-250
  • Binds to Arg and Lys sidechains
19
Q

What is Isoelectric Focusing?

A
  • No SDS needed
  • Gel matrix has pH gradient
  • Protein migrates until they are uncharged when pH = pI
20
Q

What is 2D Gel Electrophoresis?

A

Separation of proteins by pI then SDS page (sorts by molecular mass)

21
Q

What is Edman Degredation?

A
  • Improved Sanger’s protein sequencing technique
  • Polypeptide is cleaved between first and second amino acid
  • Takes ~50 min per amino acid
  • Does not work on proteins that have blocked (acetylated) N-terminus
22
Q

How does Edman Degradation work?

A
  • PITC treated with trifluoroacetic acid
  • This cleaved b/t first and second peptide
  • Thiazolinone derivative is made and the amino acid chain loses an amino acid
  • Proton is added to N
  • Process repeats ( Treat next aa with PITC and base and repeat)
  • Only works for up to 50 aa
23
Q

What is Mass Spectrometry?

A
  • Measures mass to charge ratio (m/z) of molecules
  • Can determine molecular mass of proteins
  • Can be used for aa sequencing
  • Ionizes proteins and leaves them intact
  • Two types ESI and MALDI
24
Q

What are the differences between Matrix-assisted laser ionization and Electrospray ionization?

A

Matrix-assisted laser desorption ionization (MALDI)
- Laser pulse ionizes sample in absorbing matrix
- Collision with protein and transfer change occurs
- Detector in mass spectrometer measures acceleration of ions determining their mass

Electrospray Ionization (ESI)
- Polypeptide fragments (usually tryiptic fragments which is fragments of a trypsin cleaved protein) are released via high voltage metalic capillary
- Conditions cause solvent containing peptides to evaporate
- This makes highly charged gas molecules

25
What are the advantages and disadvantages of Mass Spectrometry over Edmund degradation?
Advantages: - Takes several minutes for the whole peptide as opposed to 45 minutes per amino acid - Is not affected by blocked (acetylated) N termini Disadvantages: - Isoleucine and Leucine have identical mass - Glutamine and Lysine only differ in size by 0.036 Da
26
What is X-ray Crystallography and what are the steps?
- A method of protein structure determination based on diffraction of X-rays by protein crystals Steps: 1. Purify protein 2. Grow diffraction quality crystals 3. Determining the phases of diffracted X-rays 4. Calculate electron density map 5. Fit amino acid sequence into density map
27
What is NMR Spectroscopy, what are the steps, and what are pros and cons?
- A method of protein structure determination based on magnetic properties of several types of atoms when - Focuses on H1, N15, and C13 - Works well with proteins smaller than 100 kDa Steps: 1. Purify proteins 2. Dissolve the protein 3. Collect NMR data 4. Assign NMR signals 5. Calculate structure Pros: - No need to crystalize protein Cons: - Difficult for insoluble proteins - Works best with small proteins
28
What is Cryo-electron Microscopy?
- A form of microscopy that is good for larger protein complexes - Compiles 2D projections to generate high resolution 2D images then can be built into 3D model
29
How are antibody proteins produced?
- They are produced by B cells in the immune system - Each B cell makes a single type of antibody
30
How many and what type of chains can B cells create?
- B cells can make two classes of Ig light chains (λ and κ) and five classes of Ig heavy chains (µ, α,γ, δ, and ε)
31
What is an Epitope?
- A specific site on antigen antibodies can bind to - Example: protein from Borrelia burgodorferi causes Lyme disease and has several epitopes that antibodies can recognize
32
Define and compare monclonal and polyclonal antibodies
Monoclonal: - Homogeneous immunoglobin species that recognizes ONE epitope on an antigenic protein - Isolated from hybridomas Polyclonal: - Heterogeneous mixture of immunoglobulin proteins that recognize ONE OR MORE epitopes on an antigenic protein - Isolated from blood
33
How are polyclonal antibodies generated?
1. Immunization occurs 2. Blood sample is obtained containing nonspecific and specific antibodies 3. Purification via affinity chromatography 4. Three fractions are collected (flow through, Nonspecific antibodies, and affinity purified polyclonal antibodies
34
How are monoclonal antibodies generated?
1. Multiple immunizations 2. B cells are isolated 3. B cells are joined with immortalized tumor cell 4. Antigen specific and nonspecific hybridoma are generated 5. Antigen specific hybridoma identified via screening 6. Cloning of hybridomas 7. Tissue is cultured containing antigen specific monoclonal antibodies
35
What is Western Blotting?
- Detects proteins with high sensitivity - Used to detect proteins separated by SDS-PAGE - Uses two antibodies - Primary binds to target protein - Secondary contains enzyme for visualization
36
What is Epitope Tagging?
- Used to detect proteins when no antibody is available - Protein coding sequence of highly antigenic peptides added to sequences of cloned genes at the N and C termini forming a unique epitope which can be recognized by specific epitopes
37
What is Immunofluorescence?
- Detects proteins with high sensitivity - Identifies proteins in a cell that have treated chemically to preserve cell architecture - Can be visualized by fluorescence microscopy if the primary or secondary antibody are fluorescent or contain a fluorescent group.
38
What is ELISA?
- Enzyme linked immunoabsorbant Assay (ELISA) - Detects proteins with high sensitivity - Identifies low level antigenic proteins in solution - Used in samples such as plasma
39
What is immunoprecipitation
- Variation of affinity purification - Monoclonal antibody covalently linked to carb. bead - Incubation of protein mixture and MAb-beads which are collected by centrifugation
40
What can chymotripin cleave?
Trypsin, tryptophan, and phenol-alanine (all aromatics)
41
What can trypsin cleave?
Cleaves peptide bond at carboxyl side of Lysine and Arginine
42
What does Cyanogen bromide do?
Cleaves peptide bond on carboxyl side of Met
43
What does V8 protease do?
Cleaves peptide bond on carboxyl side of Aspartate and Glutamate
44
What can be done to counteract the limitations of Edmund degradation?
Generate small fragments by proteases such as Trysin, Chymotrypsin, Cyanogen bromide, and V8 protease

CHEM 441A+B (22 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions