Paxillin Western Blot Lab Report
Ryan Piche
BIO 410
Dr. Cooper
4/3/2013
Hypothesis: If a western blot identifying paxillin protein from zebrafish embryos raised at two
different temperatures is performed, then the embryos raised at a higher temperature will be
more developed and will have more paxillin protein present.
Figure 1. Zebrafish embryos raised at a higher temperature produce more paxillin protein
than embryos raised at a lower temperature. Zebrafish embryos were raised to an age of four
days at two different temperatures. Five embryos raised at 21 degrees Celsius and five embryos
raised at 29 degrees Celsius were placed in cultures containing 500 µL of fish water. Both
cultures were anesthetized with 50 µL of tricaine for a final anesthetic concentration of .363
mg/mL. The embryos were then boiled, broken down, and centrifuged. After, one 20 µL sample
from each culture was loaded onto an SDS-PAGE gel, along with a pre-stained protein ladder.
Above, lane L corresponds to the protein ladder, lane 1 corresponds to embryos raised at 21
degrees Celsius, and lane 2 corresponds to embryos raised at 29 degrees Celsius. Electrophoresis
was then run for 60 minutes at 165 V. A transfer apparatus was then assembled and the proteins
were transferred overnight at 50 V. After transfer, the nitrocellulose membrane was incubated at
room temperature for 5 minutes in a 2% solution of non-fat dry milk powder. Then, 1 µL of the
primary antibody (mouse anti-paxillin, 250 µg/mL at 1:1000) was mixed with 1 mL of 2% milk.
The primary antibody was added to the membrane, followed by incubation at room temperature
for 45 minutes. Following incubation, the membrane was washed three times with 1x TS. After
washing, the secondary antibody (goat anti-mouse IgG-alkaline phosphatase, Sigma, 1:5000)
was mixed with 1 mL of 2% milk. The secondary antibody was then introduced to the
membrane, followed by incubation at room temperature for 30 minutes. After the second
incubation, the membrane was washed as before. Finally, 1 mL of BCIP/NBT was added to the
membrane for protein band identification. After the protein bands darkened, the membrane was
imaged, and amounts of paxillin protein were determined and analyzed.
3
2.5
2
Relative
Amount of 1.5
Paxillin Protein
1
0.5
0
Lower Temperature
Higher Temperature
Zebrafish Embryo Treament
Figure 2. Zebrafish embryos raised at a higher temperature produced larger relative
amount of paxillin protein than embryos raised at a lower temperature. Using the
membrane image from Figure 1, relative amounts of paxillin protein were identified using
ImageJ and GIMP image manipulator software. In Figure 1, band (A) corresponds to zebrafish
embryos raised at 21 degrees Celsius, and band (B) corresponds to embryos raised at 29 degrees
Celsius. Using protein band size calculations from the ImageJ program, relative amounts of
paxillin protein were determined for both of the embryo treatments. These amounts of paxillin
protein are shown relative to the amount of protein determined for the lower temperature
treatment group.
Results
The results of the western blot show that the relative amount of paxillin for the higher
temperature embryo sample is larger than the relative amount for the lower temperature embryo
sample. This relates to our goal for this experiment: to determine whether or not the embryo
treatment of different temperatures effected the production of the paxillin protein (Figure 2).
Two distinct paxillin protein bands were identified on the membrane. At the time of analysis, it
was unknown as to why two distinct bands appeared.
Discussion
The results of the western blot have demonstrated that the zebrafish embryos raised at a
higher temperature had a larger relative amount of the paxillin protein. This is probably due to
the effect temperature has on the development of the zebrafish embryo. Specifically, paxillin is a
multi-domain protein that localizes primarily to sites of cell adhesion to the extracellular matrix
called focal adhesions. Focal adhesions form a structural link between the extracellular matrix
and the cytoskeleton (Turner 2000). Paxillin’s primary function is to act as a molecular adaptor
or scaffold protein that provides docking sites for an array of signaling and structural proteins
(Turner 2000). Paxillin binds to many proteins that are involved in effecting changes in the
organization of the actin cytoskeleton, which are necessary for cell motility events associated
with embryonic development, wound repair, and tumor metastasis (Turner 2000).
Further, studies have shown that environmental factors can have a dramatic effect on fish
growth. Specifically, these studies have demonstrated that higher temperatures promote muscle
growth in fish (Knight et al. 2011). In zebrafish, it is known that the myogenic transcription
factor MyoD is needed for muscle formation and growth. Previous studies have illustrated that
after an injury to muscle fibers in zebrafish, a regenerative process occurs (Knight et al. 2011).
After the injury, cells expressing Pax7 are recruited to the damaged zone and proliferate (Knight
et al. 2011). These cells express myogenic markers like MyoD, which help develop new muscle
fibers.
All of this demonstrates the key relationship that the paxillin protein has with wound
repair and cell growth. In the zebrafish embryos raised at a higher temperature, larger amounts of
paxillin were observed. Considering the previous studies, the embryos raised at a higher
temperature were probably more developed than the embryos raised at a lower temperature. In
this case, the larger amount of paxillin makes sense, because the protein is directly related to the
processes of growth and repair in the zebrafish embryo.
Finally, the presence of two bands on the membrane was probably due to the fact that
paxillin is a multi-domain protein. In particular, the paxillin protein has four LIM domains that it
uses to attach to specific sites in the cell (Turner 2000). These multiple domains could explain
the presence of two bands on the membrane, especially if any of the LIM protein domains are
similar in size.
Works Cited
Knight K, Leslie J, Serrana DG, Hofsten JV. Fish muscle growth and repair: models linking
biomedicine and aquaculture. J Exp Biol. 2011; 214: 2995-2996.
Turner CE. Paxillin interactions. J Cell Sci. 2000; 113: 4139-4140.