Discussion
Introduction
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Marine animals experience many changes in the
environment of their habitat.
A common change in recent years has been
ocean temperature as well as human
involvement.
When these changes occur it can cause massive
damage to the cellular function of surrounding
organisms.
In order to combat this, the organisms have
developed chaperone compounds that interact
with proteins and act as quality control.
There are two types of chaperone proteins:
Molecular and Chemical.
Molecular chaperones work by using ATP to
bind directly to proteins in order to facilitate
destruction and assist with folding. The HSP
group of chaperones uses this method. (Welch
et al., 1996)
Chemical chaperones react indirectly with
proteins in order to prevent folding proteins. This
makes them less energetically taxing.
These two are equal halves when it comes to the
internal defense mechanisms of many
organisms.
While it has been shown that marine
elasmobranches acquire chemical chaperones
through their diet (Kolhatkar et al., 2014), it has
never been proven that they are able to
synthesize them on their own.
Developing methods for the analysis of
exhaustion-exposed shark blood
Purpose
To develop and improve on methods for comparing the levels of heat shock
protein in blood, specifically that of sharks.
Materials and Methods
Samples were
taken from
Fresh caught
Blue and Mako
sharks
Gels for gel
electrophoresis were
created using standard
ingredients (See
Figure 1). Temed and
APS were added to
induce the hardening
process
Gel electrophoresis
was run for 1.5
hours before
samples were
transferred to
membranes
Membranes were
scanned and
images were
analyzed
Membranes were
then soaked in CLR
buffer in order to
activate
chemiluminescent
properties
Membranes were
placed in primary
antibody and left to
soak
Figure 4: Post Mortem Mako shark
Results
Before change
Luminol and Coumaric
Figure 2: Completed gel inside Bio
Rad Gel holder
Figure 1: From left to right:
Resolving Gel Buffer (1.5M Tris
pH 8.8), Stacking Gel Buffer (1.0M
Tris pH 6.8), 10% SDS, DDH20,
30% Acrylamide.
Hydrogen Peroxide
Temed and APS
Figure 3: Gels approximately 25% through electrophoresis
In the future, studies could be
done to validate these results as
well as confirm that they were not
obtained due to one of the
following possible errors:
• Human error
• Contamination
• Machine/Pippette error
After change
Luminol and Coumaric
are both very light
sensitive. In past studies
Luminol and Coumaric
had been reused. It was
determined that reusing
these two caused
images to be undefined
or completely blank.
When creating a CLR
buffer, 30% hydrogen
peroxide is far preferable
to 50% hydrogen
peroxide. 50% H202
creates a blurry image
and sometimes blocks it
entirely. 30% was much
clearer.
Temed and APS are chemicals that when combined create a reaction that turns gel
from a liquid to a solid. It was found that when creating gels, the APS and TEMED levels
should be increased in order to avoid leakage. The amounts must be increased evenly
in order for the hardening process to work. (For successfully completed gel see Figure
2)
Conclusion
•Using a new Luminol and
Coumaric solution, 30% Hydrogen
peroxide, and increased levels of
Temed and APS, a much more
successful gel process can be
performed.
•In previous studies many aspects
of the results were not specified.
•In the future these improved
methods will greatly help scientists
to produce brighter and more clear
images.
Bibliography
*Marshall, H., *Field, L. *Afiadata, A., Sepulveda, C., Skomal, G. & Bernal, D. (2012, in press).
Hematological indicators of stress in longline-captured sharks Comparative Biochemistry and
Physiology
Welch WJ and Brown CR. Influence of molecular and chemical chaperones on protein folding. Cell
Stress and Chaperones 1(2): 109-115.
Ashra Kolhatkar, Cayleih E. Robertson, Maria E. Thistle, A. Kurt Gamperl and Suzanne Currie
Physiological and Biochemical Zoology: Ecological and Evolutionary Approaches
Vol. 87, No. 5 (September/October 2014), pp. 652-662
Frick LH, Reina RD, Walker TI (2010a) Stress related physiological changes and post-release
survival of Port Jackson sharks (Heterodontus portusjacksoni) and gummy sharks (Mustelus
antarcticus) following gill-net and longline capture in captivity. J Exp Mar Biol Ecol 385: 29–37