Molecular Genetic Techniques
What is the difference between Differential centrifugation and rate-zonal centrifugation?
Differential centrifugation seperates mainly by mass while rate-zonal organizes by density
What are characteristics of homologous proteins?
How does Flow Cytometry sort cells?
Flow Cytometry sorts cells on basis of antibody level of fluorescence and is used primaryily to seperate white blood cells
Which type of microscope has an annular diaphragm? what function does the diaphragm serve?
Phase Contrast Microscope
provides a cone of light that is focused onto the specimen
Highly specific proteins exist in the following organelles: mitochondria, lysosome, peroxisome
Name the specific protein for each organelle
Mitochondria = cytochrome C
Lysosome = Acid Phosphatase
Peroxisome = Catalase
Match these tests with how they organize proteins (on basis of Charge, mass, Antibody, size, density)
1. Gel filtration chromatography
2. Ion-echange chromotography
3. Antibody-affinity chrtomatography
4. Gel Electrophoresis
5. SDS Page
6. Differential Centrifugation
7. Rate-zonal Centrifugation
Gel filtration chromatography - size
Ion-echange chromotography - net charge
Antibody-affinity chrtomatography - antibody
Gel Electrophoresis - charge and mass
SDS Page - mass
Differential Centrifugation - mass
Rate-zonal Centrifugation - Density
Four levels make up the hierarchical structure of a protein: primary, secondary, tertiary, and quaternary.
Provide a brief description of what makes up each level
1 (AA sequence)
2 (H-bonding and local folding)
3 (functional units or secondary interacting with one another, overall conformation)
4 (multimeric structure)
What replicates in G1 of th cell cycle
Duplication of:
RNA
Lipid
Protein synthesis
Cellular components (excluding chromosones)
Scanning Electron Microscopy
- enables viewing of metal-coated specimen (res: 10 nm)
Cryoelectron Tomography
- allows determination of 3D structure
List at least two differences in the cycles of HSP70 and HSP90
Hsp70 starts with an unfolded protein while Hsp90 starts with a partially folded protein
Hsp70 has additional proteins DnaJ/Hsp40 and GrpE/BAG1
Hsp90 has a slow step
Hsp90 is a dimeric structure, and therefore has 2 ATP binding sites
ATP hydrolysis occurs before closing on Hsp70, whereas in Hsp90 hydrolysis occurs after closing
Fill in all the blanks:
_____ and _____ are two genetic techniques that reveal mRNA expression, _____, and localization.
Microarray
In Situ Hybridization
co-regulation
_________ groups project outward frfom the surface of the helix and determine the chemical nature of helix faces. ________ usually cannot participate in hydrogen bonding and are excluded from an alpha helix
R groups project outward frfom the surface of the helix and determine the chemical nature of helix faces. Prolines usually cannot participate in hydrogen bonding and are excluded from an alpha helix
What are some features of Madin-Darby dog kidney cell culture? Why is the filter important
Porous filter on the basal lamina coated in collagen, the porous filter allows for thw cells to perform secretion function
Basal medim and apical medium
Specimen preparation for light and electron microscopy follow what four steps (list them in order)
FIX: formaldehyde
EMBED: LM = parrafin, EM = liquid plastic
SECTION: LM = 0.5- 50 mm, EM = 50 -100 nm
STAIN: H & E staining
DNA microarray analysis utilizes green, red, and yellow spots to indicate differing levels of gene expression.
Match each color to one of the following indications:
higher expression in serum positive cells
no change
higher expression in serum negative cells
green = higher expression of serum negative/starved cells
yellow = no change (equal hybridization of green and red fluorescence)
red = higher expression of serum-positive cells
If you wanted to seperate proteins on the basis of net charge, what chromatography technique would you use?
Ion-exchange chromatography
________ peptide bonds limit the shapes into which protains fold by ________ restraints and ______side chain. about _______ % of peptide bonds that have any other residue than ______ at P2 are trans configuration
Planar peptide bonds limit the shapes into which protains fold by Steric restraints and Amino Acid side chain. about 99.97% of peptide bonds that have any other residue than Proline at P2 are trans configuration
What color do these Fluorescent tags emit?
DAPI
FITC
Cy3
Cy5
Alexa 568
DAPI - blue
FITC - green
Cy3 - orange
Cy5 - red
Alexa 568 - orange/yellow
Fill in all the blanks:
_____ and _____ are two genetic techniques that reveal mRNA expression, _____, and localization.
Microarray
In situ Hybridization
co-regulation
What is the resolution power equation?
D=0.61λ /Nsina
Explain the 4 steps of western immunoblotting
1. Transfer blot/protein/SDS-PAGE to a porous membrane
2. Incubate with the primary antibody then wash (primary binds to the protein of interest)
3. Incubate with secondary antibody (whih sticks to primary Ab)
4. Detect label on Ab2 to reveal location of the protein of interest
What are the 3 alpha helix motifs and a characteristic of each?
Coiled-coil 2 a helicies wound around each other with a hydrophobic residue at 1 and 4
Helix-loop-helix uses calmodulin and is rich in glu and aspartate
Zinc-finger is a 25 residue motif with 2 invariant cysteine residues a positions 3 and 6 and 2 invariant histidine residues at positions 20 and 24
Explain FRAP process and purpose
irriversible bleaching occurs by laser in a region of interest. watch over time for restoration of fluorescence moving into the bleached portion of interest. This process shows the dynamics of molecules
Draw the stages of establishment in a cell cultre for a primary cell culture and cell line, and label the 3 stages of both
Primary cell culture
I. Increased growth rate
II. constant growth rate
III.Decreased Growth rate
Cell Line
I. Initial loss of growth
II. Senescence
III. Emergence of immortal line
Explain how the protein-protein interactions occur via FRET
Excitation of CFP via protein X emits a wavelength of 475, and when close to a YFP fluorescent (linked to another protein Y) causes YFP to emit a yellow light if X and Y proteins work together, allowing for visualization of interaction