Download Quiz II - Berkeley MCB

Name____________________________________
Quiz II
MCB 130L Summer 2007
In your research lab, your project is to characterize the function of a novel protein isolated from
the brains of Nobel laureates. You have named the protein Nobellin (encoded by the gene NobL). You
have successfully cloned the gene, expressed it as a recombinant protein, and have generated polyclonal
antibodies to the protein in mice. You are interested in seeing subcellular localization of this protein
within freshly isolated neurons.
1. (1 pt) What is the advantage of using primary cells (compared to a cell line)?
Minimal modification
2. (2 pts) Your protocol says to store your antibodies in 0.1% sodium azide as a preservative. Explain
how sodium azide might preserve your antibodies.
Inhibits respiration, prevents bacterial/fungal growth
3. (1 pt) What is the order in which you would do immunofluorescence staining of Nobellin? (Some of
these steps may be required more than once.)
__4___ mouse anti-Nobellin
__6___ FITC-rabbit anti-mouse IgG
__1___ fixation
__2___ permeabilization
__3,5__ wash & block
4.
(2 pts) Name 2 fixatives used for microscopy and the principles of fixation for each.
Aldehydes—crosslinking
Alcohols—dehydrates, precipitates proteins
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Name____________________________________
5. (2 pts) What happens if you reverse the order of fixation and permeabilization during staining of
microtubules? Is it salvageable? If so, how?
Tubulin subunits remain, fluorescence is less distinct for microtubules. Not salvageable
In order to view dynamics and determine subcellular localization of Nobellin, you decide to make a
mOrange-Nobellin fusion protein and express it in HeLa cells. mOrange is an engineered orange
fluorescent protein. Its peak excitation wavelength is 548 nm and it emits at 562 nm.
6. (2 pts) Name 2 features of a eukaryotic expression vectors are absent in bacterial expression vectors.
Eukaryotic promoter and polyA sequence
7. (1 pt) To determine subcellular localization of mOrange-Nobellin, which of the following
fluorophores (Molecular Probes) is least likely to work in colocalization studies? (Name of
fluorophore is followed by excitation/emission peaks)
(a) Alexa Fluor 532 (531/554)
(b) Alexa Fluor 546 (556/573)
(c) Alexa Fluor 555 (555/565)
(d) Alexa Fluor 568 (578/603)
(e) Alexa Fluor 594 (590/617)
(1 pts) Explain why:
Emitted wavelength is too close (562 vs. 565 nm). Filter would not be able to distinguish.
8. (2 pts) While preparing your DNA-liposome complex for transfection, the pH of your buffer is
too high, and the transfection fails. Think of two reasons why.
1) Kills cells; 2) Removes positive charge, resulting in poor uptake of liposome
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Name____________________________________
9. (5 pts) Based on data from other labs, you hypothesize that Nobellin is involved in cellular
respiration. You mutate the Nobellin homolog of yeast and find that respiration rate is lower in
the Nobellin mutant than in WT yeast. Design a simple experiment that would determine which
step in respiration is inhibited.
Flow chart—same as in respiration lab. Include glucose vs. EtOH to determine if glycolysis or
TCA cycle is affected
10. (5 pts) You discover that Nobellin can induce the transcription of several genes that result in
increased IQ. The Nobellin protein is very small, about 10 kDa and it was very effective in
clinical trials for curing stupidity. You decide to modify the protein to make it even more
effective so you add on an additional domain that adds an additional 50 kDa to the protein. When
you test your new super protein, it no longer works. What happened? How could you test your
hypothesis? How could you fix this?
In order for your cure to induce transcription it has to get into the nucleus. If after modifying it, it
no longer functions, there are several possibilities. One option is that it can no longer get into the
nucleus. This is likely because you have increased the size of your protein dramatically and it can
no longer passively diffuse through the pore. The nuclearpore allows things smaller than ~50-60
Kda to passively diffuse into the nucleus. However, bigger proteins are excluded from the nucleus
unless they contain a nuclear localizing signal. (NLS). Since you dramatically increased the size of
your protein, it most likely can no longer enter the nucleus. You can test this hypothesis by
monitoring protein localization with GFP. If this is the case, you could add an NLS to your
protein sequence and it should then be able to enter the nucleus and induce transcription.
11. (2 pts) Explain why part I (splitting of cells), but not part II (reporter gene assay) of the signal
transduction lab was done in the laminar flow hood.
Sterile technique is needed for growing more cells. Part II was the assay, where no additional cell
growth was necessary
12. (2 pts) What is the difference between a transcriptional fusion and a translational fusion? When
would a transcriptional fusion be preferable?
Transcriptional fusion: reporter fused to promoter—better for monitoring promoter strength
Translational fusion: reporter fused to gene encoding protein
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Name____________________________________
13. (2 pts) Counting Cells: Here is your hemocytometer grid of cells in Trypan Blue:
What is your concentration of cells?
13/4 x 104 = 3.25 x 104 cells/ml
Calculate the volume you need for 5 x 106 cells:
5 x 106/3.25 x 104 = 153.8 ml
Bonus (3.5 pts): Journal Club (circle the correct answer)
i.
Which is more efficient at silencing genes? (a) ssRNA (b) dsRNA
ii.
RNAi was used to silence actin-related genes in cells from: (a) C. elegans (b) Drosophila
iii.
Nuclear integral membrane protein import requires: (a) karyopherins (b) clathrin
iv.
The mitochondria signal receptor p32 was identified based on: (a) anti-idiotypic antibodies
(b) sequence homology
v.
How many olfactory receptors are estimated in rats? (a) less than 10 (b) more than 100
vi.
Where does CED-4 translocate for programmed cell death? (a) nucleus (b) mitochondria
vii.
Helicobacter pylori is the causative agent of (a) gastric ulcers (b) colon cancer
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