1. Developing methods for the analysis of exhaustion-exposed shark blood
Introduction
Discussion
Developing methods for the analysis of exhaustion-exposed shark blood
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
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.
Purpose
To develop and improve on methods for comparing the levels of heat shock protein in blood, specifically that of sharks.
Materials and Methods
Gels for gel electrophoresis were created using standard ingredients (See Figure 1). Temed and APS were added to induce the hardening process
Samples were taken from Fresh caught Blue and Mako sharks
Gel electrophoresis was run for 1.5 hours before samples were transferred to membranes
Membranes were then soaked in CLR buffer in order to activate chemiluminescent properties
Membranes were scanned and images were analyzed
Membranes were placed in primary antibody and left to soak
Figure 4: Post Mortem Mako shark
Results
Before change
After change
Conclusion
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.
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.
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.
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.
Hydrogen Peroxide
Bibliography
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)
*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):
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
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
Temed and APS
Figure 3: Gels approximately 25% through electrophoresis