AN EVALUATION OF EXTRACTED PROTEINS FROM BEAN BEETLE B. Suarez, R. Martinez, O. Diaz, H. Jones, T. Ashraf, E. Priddis, K. Durham, Undergraduate Biology Research, Cochise Community College, Sierra Vista, AZ

INTRODUCTION Genomic and proteomic studies can reveal multi-dimensional aspects of biological model organisms. DNA sequencing and short tandem repeats are utilized to characterize organism’s phylogenetic relationships; another approach is to study their various proteins. Many genomic studies utilize extraction and amplification of nucleic acids to help make detection more straightforward. There is no proteomic procedure similar to PCR that would identify proteins at their naturally existing concentration, as well as the presence of many other proteins for comparative studies. Most methods for studying proteins revolve around running 1D, 2D, or 3D gels, and comparing and identifying similar proteins.  The proteomics approach is valuable when there is very little known about the genome of an organism. The gene is a blue print for making proteins; hence, a study of an organism’s proteins can give insight to the expression of its genes.  

BACKGROUND INFORMATIONBean beetles (Callosobruchus maculatus) are an agricultural pest that has a limited life span of two to three weeks. The larvae of this species feed and develop entirely on the seeds of legumes, hence the name bean beetle. The adults do not need food or water, and spend their limited lifespan mating and laying eggs on beans.

Once mated, adult females will lay a single fertilized egg on the exterior of a bean. Individual eggs are small (visible with the naked eye), and appear to be clear, shiny and firmly glued to the bean surface. Larvae will burrow into the bean's center and develop into a pupa after hatching. Later, the pupa undergoes metamorphosis to an adult and emerges through the seed coat as an adult beetle (winged or non-winged). The adults are fully mature 24 to 36 hours after emergence. Adult males and females then proceed to mate and lay eggs for the next generation. Studies have shown that there are a total of seven larval stages in the development of a bean beetle.

Adult male and female bean beetles are easily distinguished from one another by overall appearance. The most distinguishing characteristic is the pattern on the abdominal plate covering. In the female, the body is enlarged and is darkly colored on both sides. In the male, the body is smaller, lacks stripes, and is slightly lighter in pigmentation. Because of the ease of raising bean beetles in captivity, as well as the ability to easily distinguish sex, bean beetles are being utilized as model organisms in classroom environments.

C. VERTICAL GEL SDS - PAGE ELECTROPHORESIS: Equal amounts of adult male and female bean beetle samples were loaded into the gel cassette, including two wells of a standard protein ladder for comparison. The gel was run using Tris glycine/SDS buffer at 200 volts. Coomassie blue stain was used to stain the gel and the gel was kept in destaining solution over night.

D. MOLECULAR WEIGHT ESTIMATION USING LOGGER Pro: A photo of the gel was taken with a digital camera and uploaded into Logger Pro. A line of best fit was created and the molecular weight of each band was calculated in comparison to the standard protein ladder.

RESULTSA representative graph of the BSA protein standards is shown in figure 1. The absorption was obtained from the spectrophotometer and graphed against the protein concentration. Figure 2 is the image of the gel electrophoresis results with the sample and ladder proteins. The precast gel cassette has 10 wells, 8 of which were loaded for protein samples while lanes 9 and 10 were loaded with a protein standard ladder. Lanes 1, 2, 5, and 6 can be ignored, as they did not stain well enough to display the dark bands characteristic of separating proteins. The band profiles of lanes 3, 4, 7, and 8 do, however, indicate the presence of distinct proteins. Figure 3 represents the line of best fit constructed by the distance traveled by each protein. The distance traveled by the sample proteins were extrapolated from this line. 

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and new words". Electrophoresis 19 (11): 1853–61.Beck, C., Blumer, L., Bean Beetle_A Model Organism for Inquiry-based Undergraduate Laboratories, 9

September 2013. Web: http://www.beanbeetles.org/; Blackstock, W.P., Weir, M.P. (1999). "Proteomics: quantitative and physical mapping of cellular proteins". Trends

Biotechnol. 17 (3): 121–7.Cilia, M., Fish, T., Yang, X., McLaughlin, M., Thannhauser, T.W., Gray, S. (2009). “A comparison of protein

extraction methods suitable for gel-based proteomic studies of aphid proteins”. Journal of Biomolecular Techiques 20 (4): 201-15.

ACKNOWLEDGMENTSHewlett, J., Bock, H., Tiberio, M., Community College Undergraduate Biology, Research Initiative (CCURI), Finger Lakes Community College. Anderson, N., Ph.D., Director of the BIOTECH Project, University of Arizona.Beck, C., Ph.D., Professor of Pedagogy, Department of Biology, Emory University,.Blumer, L., Ph.D., Professor, Department of Biology, Morehouse College.

Cochise College: Rottweiler, J.D., President Fick, V., Vice President of Instruction Krueger, B., Dean Math and Science Nuetzel, D., Administrative Assistant Math and Science Bambi Randall, Science Lab Technician Cabello, C., Chemistry Instructor Woodrow, A., Videographer Instructional Media Specialist

PROCEDUREA. PREPARE SAMPLE FOR ASSAY: Twenty adult male and twenty adult female beetles were homogenized in separate Eppendorf tubes with 100 µl Laemmli Sample Buffer. The samples were kept at room temperature for five minutes and then centrifuged for twenty minutes at 12000 rpm. ~50 µl of the supernatant was aspirated into a fresh tube. The tube was placed in a 95°C heat block for five minutes.  B. DETERMINE PROTEIN CONCENTRATION OF SAMPLES: The extracted protein samples were compared with the protein standards (2, 4, 6, 8, and 10 mg Bovine Serum Albumin (BSA/ml)) using a spot test on Whatman paper. The samples were then stained with Coomassie blue for thirty minutes, and then destained for an hour. The amount of Coomassie stain bound to each protein is proportional to the amount of protein in that spot. All samples were then soaked overnight in 2% SDS buffer. The total absorption for each bean beetle sample was determined using a spectrophotometer at 590 nm. The protein standards’ concentrations were graphed against their absorptions and the best-fit line was determined. Using the slope-intercept of this line, the protein concentration for each bean beetle sample was calculated based on its corresponding absorption.

FUTUREContinuing studies for this project include extracting and determining protein concentrations from the larval stage of Callosobruchus maculatus. The focus would then be to compare extracted larval proteins with results from extraction of adult proteins. Since all metabolic activity in the adult bean beetle relies solely on the nutrients collected in the larval stage, it is hypothesized that the proteins that are involved in consumption of the bean will be discovered through a comparison using Vertical Gel SDS-PAGE Electrophoresis. Furthermore, through molecular mass identification of these proteins, as well as DNA technology, the genes coding for bean consumption can be manipulated for pest control or used for developing plant immunity.

DISCUSSION From Figure 1, the linear relationship of absorption compared to protein concentration was determined. The best-fit y-intercept equation was used to determine the protein concentration of the various samples based on their absorption. The proteins for the adult male and female samples were extracted in sodium dodecyl sulfate (SDS), a negatively charged molecule which binds to proteins every 2 – 3 amino acids, modifies the tertiary protein structure, and changes the overall charge to a negative state. This negative charge is directly proportional to the protein’s molecular mass. SDS also denatures the proteins and allows proteins to separate based on their molecular mass-to-charge ratio. To assess the relative molecular weights of these proteins, samples were run alongside the known Precision Plus protein molecular ladder from Bio Rad in a Vertical Gel SDS-PAGE Electrophoresis. With the influence of a constant electrical current at 200V, smaller molecular proteins travel a farther distance down the gel than the larger molecular proteins do, creating the appearance of distinct bands. In evaluating bands formed by proteins found in male and female bean beetle samples (Table 1), there seems to be no significant difference in pattern.

Lanes 1, 2 , 5, and 6 did not stain well, due to the experimental use of samples containing a lower concentration of bean beetle protein. Thus, their results were not used for analysis. The dominant bands on lanes 3, 4, 7, and 8 seem to be of similar molecular weights. The expectation was to find some variation with one or more of the protein bands. Since this was not found, the current study suggests that similar molecular mass proteins are present in both sexes of the adult bean beetle. Perhaps these proteins are associated with membranes such as the plasma membrane, or organelle membranes from ribosomes or endoplasmic reticulum, which are common to both sexes.

Table 1. Comparison of adult male and female proteins against a standard protein ladder.

Figure 2. Protein bands seen in male and female adult against standard protein ladder.

Figure 3. Estimated molecular mass of proteins using Logger Pro.

Figure 1. Protein absorption graphed against the known protein concentrations of BSA standards.

ABSTRACT In this study, proteins were extracted using a simple homogenization method. Protein concentrations from samples of male and female adult bean beetles (Callosobruchus maculatus) were determined through comparison with protein standards (2, 4, 6, 8, and 10 mg Bovine Serum Albumin (BSA)/ml) after performing a spot test. Protein absorption was determined for each of the standards and all bean beetle samples using the spectrophotometer at 590 nm. The protein standards were graphed against their corresponding absorptions so that a best-fit line for all protein samples could be determined. Using the slope intercept form of the best-fit line, the protein concentration of each of the bean beetle samples was calculated. The values obtained were used to load an equal amount of protein in the Vertical Gel SDS-PAGE Electrophoresis for subsequent protein identification. The resultant gel was photographed and the image inserted into Logger Pro to create a standard curve and calculate the molecular weight of each experimental protein band through comparison against a standard protein ladder.