Exercises for BMC I
08 December 2014
In each case, please justify briefly your answers!
1) Please go to http://www.ncbi.nlm.nih.gov/protein/CAA38458.1 to copy and
paste the amino acid sequence of the Drosophila Frizzled protein. Use
SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1) to
determine if this protein is likely to be nuclear.
(Answer: no, there are 7 predicted transmembrane segments –thus this is likely to be a
transmembrane protein, which is indeed the case: Wnt co-receptor)
2) You analyze a novel protein called Poppy. You raise an antibody against
Poppy and obtain the following result by Western blot analysis of lysates from
cells in mitosis (lane 1) or in the G1 phase of the cell cycle (lane 2). Why do
you think the same protein migrates differently in the two lanes? How would
you test your hypothesis?
(Answer: this likely reflects a post-translational modification, for instance
phosphorylation by Cdk1, since the lysate loaded in lane 1 is from mitotic cells –they
should be able to find this out; you could then test this hypothesis by adding a
phosphatase to the mitotic extract)
3) You suspect that Poppy is phosphorylated in neurons. Which of the following
methods can you use to test this hypothesis? [from a previous exam…]
a) Fluorescence-activated cell sorter (FACS)
b) Ultracentrifugation
c) Mass spectrometry
d) X-ray crystallography
e) Yeast two-hybrid
(Answer: c)
4) You analyze Poppy, which has a size of 75 kDa (kiloDaltons) and its interacting
partner protein Seed, which is 150 kDa and in the same complex as Poppy. Which of
the following methods cannot distinguish these two proteins?
a) SDS-PAGE
b) TAP-tag purification
c) 2D gel analysis
d) Mass-spectrometry
e) Western blot
(Answer: b)
5) Which of the following experimental approaches can help you identify novel partner
proteins that associate with the Poppy protein?
chemical biology, 2D polyacrylamide-gel electrophoresis, yeast two-hybrid, mass
spectrometry, FCS
(Answer: yeast two-hybrid, mass spectrometry)
6) You clone the cDNA encoding Poppy in front of the cDNA encoding GFP,
and discover that the resulting fusion protein is present in the nucleus as well
as in the cytoplasm. Can you imagine an experiment that could allow you to
test if the nuclear and cytoplasmic pools of GFP-Poppy exchange with one
another?
(Answer: FRAP –i.e. you can photobleach the nuclear pool and ask whether there will
be recovery of fluorescence in the nucleus thereafter –and loss of fluorescence from the
cytoplasm, and reciprocally)