Lecture 2: Tools of Cell Biology Flashcards
Most Biochemical analysis of cell function requires ___ amounts of material
large -can come from whole tissues and organs, or from cultured cells
Most animals cells require a ____ to grow on and the appropriate mixture of ___.
surface, nutrients and growth factors
Primary cultures
-Cells isolated directly from a particular tissue • Neurons, myobloasts, adrenal chromaffin cells etc – Usually do not survive many passages in culture – They age, and die in a process called replicative senescence
Cell Lines
-Most commonly used cells lines have been modified to prevent death in culture – This is usually the result of alterations in normal cell cycle control mechanisms such as those that occur during cancer – These cell lines are referred to as “transformed cell lines” and grow in culture indefinitely and can be frozen and revived.
Cell fractionation
• Cells can be separated into their component fractions • Cell extracts provide an accessible system to study cell functions
____ can be used to fractionate cells
differential centrifugation
What speeds do you spin to get which organelles?
10,000 g for 10 mins= Whole cell, nuclei, and cytoskeleton
20,000 g for 20 mins= mitochondria, lysosomes, peroxisomes
80-100,000 g for 1 hr= microsomes, small vesicles
150-200,000 g for 3 hrs= ribosomes, viruses, large macromolecules
Density Sedimentation
a technique used to separate molecules on the basis of buoyant density.
____ Chromatography Exploits Specific Binding Sites on Proteins
Affinity
____ Provide an Easy Way to Purify Proteins
Genetically-Engineered Tags
Purified ___ Systems are Required for the Precise Dissection of Molecular Functions
Cell-Free
What are three types of chromatography? How do they work?
What are the common epitote tags and how do they work?
• GST, MBP, His-tag, V5, Flag, Myc, HA
Affinity purification approaches to study protein complexes
- Immunoprecipitation
- Glutathione-S-Transferase (GST) pull down
What is GST?
Sodium dodecyl sulfate (SDS)
a powerful ionic detergent
Method of separation by SDS-PAGE
• Separation based on
charge to mass ratio
• Since the protein is
(usually) uniformly
coated with SDS,
separation is largely
based on mass (and
shape)
How does isoelectric focusing work?
What are two ways to analyze protein structure?
X-ray crystallography and NMR
X-ray crystallography
x-ray diffraction pattern obtained from a protein crystal
NMR spectroscopy
Nuclear Magnetic Resonance spectroscopy-The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged. If an external magnetic field is applied, an energy transfer is possible between the base energy to a higher energy level (generally a single energy gap). The energy transfer takes place at a wavelength that corresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to yield an NMR spectrum for the nucleus concerned.
Northern Blot
RNA
Southern blot
DNA
Western Blot
proteins
DNA is cut with…
restriction endonucleases
Restriction digested DNA is annealed by….
Ligases using ATP, DNA polymerase fills in any gaps
PCR
The polymerase chain reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
-Denaturing with heat, cool to anneal primers, elongation
Use of PCR to amplify a genomic or cDNA clone
How to produce recombinant protein
