Zaidi: Analyzing Cell, Molecules and Systems 1 Flashcards
Primary cell culture
Derived directly from animal
Usually survive for a finite period of time
Established or continuous cell lines
Primary culture that has been made immortal
Sub-cellular fractionation
Reduces complexity of material
Centrifugation
Separate different cell types based on size/density
Ultracentrifugation
Separate organelles
Lysis of synaptosomes gives
Synaptic plasma membrane
What is enriched in synaptic plasma membrane
Calcium and ATPase
Recombinant DNA technology use
Used to overexpress a protein to purify it more easily
PMCA activity inhibited by
Reactive oxygen species
Techniques used to analyze proteins
SDS-PAGE
Western blotting
ELISA
Mass spectrometry
SDS-PAGE used to
Analyze unkown proteins
Can visualize separated proteins with stains/dyes
SDS is
Largely hydrophobic with a negative charge
- Unfold proteins
- Gives all proteins uniform charge
Beta-mercaptoethanol
Present in SDS-PAGE
Reduces disulfides/further denatures protein
Western blotting/immunoblotting
Analyzing specific/known proteins
Western blotting technique
Resolve protein samples on PAGE
Transfer fractionated proteins from gel onto membrane
Block membrane with neutral protein
Incubate membrane with HRP-labeled antibody
Incubate blot with chemiluminescent HRP substrate and expose to film
Western blotting antibody explanation
Introduce primary antibody to antigen in question
Introduce secondary antibody with markers
ELISA
Tests for the levels of specific antigen or antibody concentrations using corresponding antigen or antibody
ELISA simple breakdown
Probe patient serum with either test antibody or antigen
Antigen/antibody is conjugated to reactive (color-changing) enzyme
Antigen/antibody presents with color change
Indirect ELISA purpose
Measure the amount of an antibody in a samle
Sandwich ELISA purpose
Measure the amount of an antigen in a sample
Indirect ELISA process
Start with antigen coated well
Specific antibody binds antigen
Enzyme-linked antibody added-binds to specific antibody
Substrate is added, enzyme causes color change, rate of color change is proportional to amount of specific antibody
Sandwich ELISA process
Monoclonal antibody coated well
Antigen binds to antibody
Second monoclonal antibody w/enzyme binds to immobilized antigen
Substrate added, enzyme causes color change, rate of color change is proportional to amount of antigen
Mass spectrometry used to
Identify unknown proteins
Mass spectrometry requires
Tryptic digestion products (peptide fragments)
Ionization (charge)
Detection method
Computer databases with known protein sizes
