Analyzing Cells, Molecules, And Systems 1 Flashcards
Discerning protein size in SDS-PAGE
The higher up on the paper, the LARGER the Protein is
- measured in daltons
Established/Continuous Cell lines
- Primary culture that has been made IMMORTAL (infinite) by. Transformation
- Most commonly TUMOR derived
(Or Virus transformed)
What are proteins treated with BEFORE they enter the chambers in SDS-PAGE?
- SDS
- To give proteins an overall NEGATIVE charge - β-Μercapto-Ethanol
- Reduces DISULFIDES
- Denature
Sub-Cellular Fractionation:
Tissue
Mechanical Blending
Sub-Cellular Fractionation:
Centrifugation
- To separate different cell types
- Based on SIZE
Application of cell culturing
- Production of biological products
- Testing of drugs
- Gene analysis
- Regenerative medicine
The different matrices for Column Chromatography
- Ion-Exchange
- Positive bead binds Negative molecule - Gel-Filtration
- Small molecules get trapped
- Large molecules elute out - Affinity
- Enzymes bind COVALENTLY to substrate bead
Sub-Cellular Fractionation:
Ultracentrifugation
Separate organelles
SDS-Page Characteristics
- 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
Purpose of Western Blotting?
- Analyzing of SPECIFIC/Known protein
- Uses Primary And Secondary Antibodies
Indirect ELISA Characteristics
- 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
Examples of established of continuous cell lines
- Chinese hamster ovary
- Human neuroblastoma
- Human embryonic Kidney
Before you can do protein purification, you usually need to _____ first. Why?
Sub-cellular Fractionation
- Reduces the complexity of the material
Sub-Cellular Fractionation:
Homogenate
SUSPENSION of different cell types
Sub-Cellular Fractionation:
Lysis of cell
- Osmotic Shock, ultrasonic vibration, mechanical blending, forcing through small orifice
Primary cell Cultures
- 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
What is mass spectroscopy used for?
- ID UNKNOWN Proteins
Requires:
- Tryptophan Digestion products (Fragments)
- Ionization (Charge)
- Detection Method
- Computer Database with KNOWN protein size
Cell Fractionation by centrifugation:
Pellet order by increasing speed
- Whole cell, Nuclei, and Cytoskeleton
- Mitochondria, Lysosomes, and Peroxisomes
- Microsomes and Small vesicles
- Ribosomes, viruses, and large macromolecules
Purpose of ELISA
- Tests the levels of Specific [ANTGEN] or [AB]
Two types of ELISA used
- Indirect
- Measures the amount of an AB in a sample - Sandwich
- Measures the amount of an ANTIGEN in the sample
Two types of Ultracentrifuge
- Fixed angle Rotor
2. Swinging Bucket Rotor
Examples of Primary Cell culture
- Primary NEURONS
2. Cardiomyocytes
Cell culture overview
- Isolate from tissue
- Revive frozen cell population
- Grow in culture using media
- Plate on appropriate surface (Sub-Culture)
- Count cells
- Cryopreservation
Sandwhich ELISA Characteristics
- 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
