Transcript Document

Research Experience in Molecular Biotechnology & Genomics
Summer 2010
Center for Integrated Animal Genomics
Lucas Hemmer1, Changfu Yao2, Kristen M. Johansen2
1Nebraska
Wesleyan University, Lincoln, NE 2Department of Biochemistry, Biophysics, and Molecular Biology, Ames, IA
Affinity Purification of GFP Antibody and Observations of Chromator during Mitosis
in Drosophila melanogaster
Abstract
The mitotic spindle is thought to be the primary structure
supporting mitosis in eukaryotic organisms and consists of
polymerized tubulin enacted upon by motor and non-motor
proteins. The intricacies of its function have yet to be explained. A
hypothetical system of proteins, known as the spindle matrix, has
been proposed to explain the force required for mitotic spindle
functions. Several proteins have been identified in Drosophila to
be part of this spindle matrix. Of particular interest is Chromator,
which has yet to be tested for persistence in the event of mitotic
spindle disassembly, which is one of the predictions of the
hypothetical spindle matrix. Drosophila embryos were collected,
fixed, stained, and observed under a fluorescent microscope.
Control stainings with anti-Skeletor and –Chromator antibodies
revealed spindle-like staining that co-incided with the microtubule
spindle. However, the mitotic spindle failed to depolymerize using
the known microtubule destabilizing
nocodazole, leaving
inconclusive observations of Chromator’s behavior. A possible
problem with nocodazole storage likely reduced its effectiveness
during treatment, and thus more observations will be necessary to
determine if Chromator stability is based on mitotic spindle
stability. In a second project, the GFP protein’s unique fluorescent
properties are used in many similar protein tagging research. A
GFP antibody is useful in detecting the protein as well as
revitalizing fluorescence. The current GFP-Ab was tainted with
nonspecific background which interferes with effectiveness. Stock
GFP-Ab was affinity purified and concentrated while being
analyzed with spot tests and Western blots. Results show that
GFP-Ab was successfully purified for later use.
Results
NOCODAZOLE TREATMENT
•GFP-Ab Affinity Purification. GFP-GST complex was
extracted from transformed competent cells (pGEX-4T-2GFP) and eluted from glutathione beads. The antibody was
concentrated by ammonium sulfate, dialyzed, and purified by
an Affinity Column. Spot Tests and Western Blots tested
increased concentration and purity.
•Embryo Collection and Staining. 3 hr embryos were
dechorionated and fixed with 1:1 heptane/Bouin’s fluid,
stained with primary and secondary antibodies and viewed
under a fluorescent microscope.
•Microtubule Depolymerization Experment. After
dechorionation, the embryos were placed in heptane with 10
μM nocodazole and shaken for 1.5 min before addition of
Bouin’s fluid.
CONTROL
Methods
B
Affinity Purified
1:1
1:5
1:50
Control
1:5
1:50
1:100
1:1
1:3000
1:100 1:500
1:100
A
1:100
1:500 1:1000
Skeletor
DNA
Composite
B
Tubulin
Chromator
DNA
Composite
Skeletor
DNA
Composite
Skeletor
DNA
Composite
C
Tubulin
D Chromator
Discussion/Conclusions
Figure 1: Isolation of GFP-GST complex from transformed
E. coli. A: Small induction for GFP-GST expression.
B: GFP-GST elutions from a glutathione bead column.
Introduction
Tubulin
Figure 4: Fluorescent microscopy pictures of a single
embryonic nucleus in metaphase. Primary antibodies
used include: anti-α-tubulin (green) or IgM-tubulin (red) for
tubulin, 1A1 for Skeletor, 6H11 for Chromator, and
Hoechst for DNA.
GFPGST
A
A
1:1000
1:3000
B
Figure 2: Spot tests of anti-GFP with dilution
concentrations. A: Prior to concentration. B: After being
concentrated with ammonium sulfate and dialyzed in PBS.
•Spot tests indicate that affinity purification diluted anti-GFP,
once treated with ammonium sulfate and dialyzed the
antibody was more concentrated than the commercial stock
•Western Blot confirms that affinity-purified GFP-Ab has
signficantly reduced background as compared to the
commercial stock and looks to perform more efficiently in later
laboratory procedures
•The original nocodazole treatment failed to depolymerize the
mitotic spindle completely. Further investigation revealed the
expiration of stock supply likely to blame.
•The reduction of Chromator staining observed after partial
microtubule depletion induced by nocodazole treatment could
be consistant with the hypothesis that Chromator is
dependent on the mitotic spindle for stability. More studies
must be made under successful conditions to rest on a more
solid conclusion
References
•Chromator is one of several spindle matrix proteins discovered in
Drosophila melanogaster, an exceptional model organism
•Properties of hypothetical spindle matrix proteins include: 1) A
fusiform matrix structure 2) Persistence in absence of microtubules
3) Perturbation of matrix would affect mitotic assembly and or
function 4) Interaction with the microtubules and/or motor proteins
•Chromator interacts directly with microtubules, raising the
question of whether it depends on microtubules for stability
•Green fluorescent protein (GFP) extracted from Auquorea victoria
is used extensively in biochemical and cell biology; its unique
fluorescent properties are used for protein tagging and gene
expression
•GFP antibodies can detect GFP and revitalize fluorescence in
fixed preparations using secondary antibodies,
immunocytochemistry or be used for Western Blot detection
•The current stock anti-GFP retains nonspecific background that
reduces its efficiency in laboratory techniques
AntiGFP
Commercial
anti-GFP
Antitubulin
Figure 3: Western blot of NCD-GFP fusion protein detected
with affinity purfied anti-GFP with anti-tubulin as the control
and dilution ratios above.
Acknowledgements
I would like to thank my mentor, Dr. Kristen M. Johansen, and her
collaborator, Dr. Jørgen Johansen, for letting me use their lab. I
would also like to thank Changfu Yao for his help with my project as
well as the other members of the laboratory for support. Finally, I
thank the National Science Foundation, Iowa State University, and
especially Dr. Max Rothschild and Justin Rice for putting together
this REU program and allowing me this opportunity to perform
research.
•Ding Y, Yao C, Lince-Faria M, Rath U, Cai W, Maiato H, Girton J,
Johansen KM, Johansen J. 2009. Chromator is required for proper
microtubule spindle formations and mitosis in Drosophila.
Developmental Biology 334: 253-263.
•Johansen J, Johansen KM. 2009. The spindle matrix through the cell
cycle in Drosophila. Fly 3:3, 1-8.
•Johansen KM, Johansen J. 2007. Cell and molecular biology of the
spindle matrix.
International Review of Cytology 263: 157-207
•Johansen KM, Johansen J. 2003. Studying nuclear organization in
embryos using antibody tools. Methods in Molecular Biology 247: 215234.
•Qi H, Rath U, Wang D, Xu Y, Ding Y, Zhang W, Blacketer MJ, Paddy
MR, Girton J, Johansen J, Johansen KM. 2004. Megator, an essential
coiled-coil protein that localizes to the putative spindle matrix during
mitosis in Drosophila. Molecular Biology of the Cell 15: 4854-4865.
•Rath U, Wang D, Ding Y, Xu Y, Qi H, Blacketer MJ, Girton J, Johansen
J, Johansen KM. 2004. Chromator, a novel and essential
chromodomain protein interacts directly with the putative spindle matrix
protein Skeletor. Journal of Cellular Biochemistry 93: 1033-1047.
•Tsien RY. 1998. The Green Fluorescent Protein. Annual Review of
Biochemistry 67:509-544.
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371