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Molecular Biology Exam 2 > Analyzing Cells, Molecules, And Systems 1 > Flashcards

Analyzing Cells, Molecules, And Systems 1 Flashcards

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1
Q

Discerning protein size in SDS-PAGE

A

The higher up on the paper, the LARGER the Protein is

- measured in daltons

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2
Q

Established/Continuous Cell lines

A
  • Primary culture that has been made IMMORTAL (infinite) by. Transformation
  • Most commonly TUMOR derived
    (Or Virus transformed)
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3
Q

What are proteins treated with BEFORE they enter the chambers in SDS-PAGE?

A
  1. SDS
    - To give proteins an overall NEGATIVE charge
  2. β-Μercapto-Ethanol
    - Reduces DISULFIDES
    - Denature
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4
Q

Sub-Cellular Fractionation:

Tissue

A

Mechanical Blending

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5
Q

Sub-Cellular Fractionation:

Centrifugation

A
  • To separate different cell types

- Based on SIZE

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6
Q

Application of cell culturing

A
  1. Production of biological products
  2. Testing of drugs
  3. Gene analysis
  4. Regenerative medicine
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7
Q

The different matrices for Column Chromatography

A
  1. Ion-Exchange
    - Positive bead binds Negative molecule
  2. Gel-Filtration
    - Small molecules get trapped
    - Large molecules elute out
  3. Affinity
    - Enzymes bind COVALENTLY to substrate bead
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8
Q

Sub-Cellular Fractionation:

Ultracentrifugation

A

Separate organelles

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9
Q

SDS-Page Characteristics

A
  • Largely Hydrophobic w/ a negative charge
  • Unfolds proteins
  • Gives it a uniform charge (Negative)
  • Allows for ALL protein to migrate toward a POSITIVE charge in the presence of a current
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10
Q

Purpose of Western Blotting?

A
  • Analyzing of SPECIFIC/Known protein

- Uses Primary And Secondary Antibodies

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11
Q

Indirect ELISA Characteristics

A
  • Amount of AB in sample
  • Uses well coated with AG
  • Specific AB bings to AG
  • Enzyme AB binds to Spec. AB
  • Substrate added and Converted by enzyme to color
  • Rate of color formation is proportional to the amount of specific AB
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12
Q

Examples of established of continuous cell lines

A
  • Chinese hamster ovary
  • Human neuroblastoma
  • Human embryonic Kidney
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13
Q

Before you can do protein purification, you usually need to _____ first. Why?

A

Sub-cellular Fractionation

  • Reduces the complexity of the material
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14
Q

Sub-Cellular Fractionation:

Homogenate

A

SUSPENSION of different cell types

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15
Q

Sub-Cellular Fractionation:

Lysis of cell

A
  • Osmotic Shock, ultrasonic vibration, mechanical blending, forcing through small orifice
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16
Q

Primary cell Cultures

A
  • Derived DIRECTLY from the animal
  • Involves enzymes to isolate the cell of interest
    (From a heterogenous population)
  • Usually survive for a FINITE period of time
17
Q

What is mass spectroscopy used for?

A
  • ID UNKNOWN Proteins

Requires:
- Tryptophan Digestion products (Fragments)

  • Ionization (Charge)
  • Detection Method
  • Computer Database with KNOWN protein size
18
Q

Cell Fractionation by centrifugation:

Pellet order by increasing speed

A
  1. Whole cell, Nuclei, and Cytoskeleton
  2. Mitochondria, Lysosomes, and Peroxisomes
  3. Microsomes and Small vesicles
  4. Ribosomes, viruses, and large macromolecules
19
Q

Purpose of ELISA

A
  • Tests the levels of Specific [ANTGEN] or [AB]
20
Q

Two types of ELISA used

A
  1. Indirect
    - Measures the amount of an AB in a sample
  2. Sandwich
    - Measures the amount of an ANTIGEN in the sample
21
Q

Two types of Ultracentrifuge

A
  1. Fixed angle Rotor

2. Swinging Bucket Rotor

22
Q

Examples of Primary Cell culture

A
  1. Primary NEURONS

2. Cardiomyocytes

23
Q

Cell culture overview

A
  1. Isolate from tissue
  2. Revive frozen cell population
  3. Grow in culture using media
  4. Plate on appropriate surface (Sub-Culture)
  5. Count cells
  6. Cryopreservation
24
Q

Sandwhich ELISA Characteristics

A
  • Amount of Monoclonal AG in sample
  • Uses well coated with AB
  • AG bings to Monoclonal AB
  • Enzyme AB binds to AG on MCAB
    (Forming a sandwich)
  • Substrate added and Converted by enzyme to color
  • Rate of color formation is proportional to the amount of specific AB
25
Q

Purpose of SDS-Page

A

Separation of Proteins

Molecular Biology Exam 2 (7 decks)

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