Unit 1 Flashcards
What are two major groups of identifying proteins?
(1) protein labeling
(2) protein tracking
What are four examples of protein labeling?
(1) radioactivity
(2) fluorescence
(3) chemical tags (His Myc)
(4) chemical cross linking
How are protein tracking v. protein labeling distinguished?
protein labeling - tell different types of proteins apart
protein tracking - find where protein is
what are five strategies of protein tracking?
visual (1) FACS (2) microscopy biochemical (3) size (4) charge (5) enzymatic assays
what are four ways of protein size separation?
(1) SDS page
(2) liquid gel chromatography
(3) differential centrifugation
(4) density grade centrifugation
what are compartmental enzymes?
all of the closed parts within the cytosol of a eukaryotic cell, usually surrounded by a single or double lipid layer membrane. These compartments are often, but not always, defined as membrane enclosed regions. The formation of cellular compartments is called compartmentalization.
what are three membrane bound organelles involved in protein processing
(1) nucleus
(2) ER
(3) Golgi appartus
what are four other organelles?
(1) peroxisome
(2) endosome
(3) lysosome
(4) vacuoles
what is the function of the smooth ER?
lipid generation and detoxification of hydrophobic chemicals
what is the function of the rough ER?
protein synthesis, folding and quality control (including degradation)
what are two other functions of the ER
(1) lipid donation to other organelles
(2) Ca2+ homeostasis
What is the purpose/strength of the Western blot?
(1) use specific Abs to probe for P of interest after SDS-PAGE
(2) very common technique to determine presence of specific P
What are two weakness of Western blot?
(1) background depending on Ab
(2) follows SDS-PAGE, time comsuming
What is the purpose/strength of the IP?
(1) clean WB by pre-selection
(2) co-IP proteins bound with IP to be collected and rung on SDS page
Brightfield MS?
not very useful for in depth question
Fluorescent MS, pros and cons?
(+) useful in determining where P is
(-) very difficult determine exactly which compartment that P is in
EM MS, pros and cons?
(++) even more useful in telling where P is
(-) long time, difficult,cyro even harder
How can unstained living cells be visualized?
phase contrast microscope and DIC differential interference constrast
What is phase contrast microscopy primary used for and what no?
examining location and movement of larger organelles
works for single cells and thin layers but not thick layers
What two additional parts does phase contrast microscope have to work?
(1) phase plate in the objective
(2) annular diaphragm
What can DIC do?
see extremely small details and thick objects
What is typical for preparing specimens to be observed with light microscope?
(1) fix with chemical cross linking
(2) embed on paraffin or plastic
(2) section with mircotome
(3) stain with chemical dye
What are steps to prepare sample for observation by fluorescent microscope?
(1) chemically fix and make permeable
(2) incubate primary Ab and wash
(3) incubate with secondary Ab and wash
(4) mount on specialized mounting medium
How does DIC work?
gradient of refractive index of two beams across speciman
How does phase contrast work?
variations in thickness or thinness across speciman
What does DIC stand for?
differential interference contrast
