AcknowledgmentsI would like to thank Dr. Zies for supervising me and aiding me

throughout the duration of this research project.

AbstractIn the fall of 2014, I was enrolled in a biology department research

intensive course where my partners and I carried out a research project. The goal of the project was to study the expression of the CIN8 protein during the yeast cell cycle. The function of CIN8 is to assemble the spindle fibers and control their function during anaphase. We therefore hypothesized that its expression would be the highest during anaphase and lowest during prophase of the cell cycle. Although we were able to isolate protein from different points in the cell cycle, we were unable to successfully visualize the CIN8 protein using a Western blot technique. This spring, I enrolled in URES 197. My goal was to trouble shoot the protocols for the various steps of this project. These techniques include isolation of total protein from yeast, measuring protein concentration using Pierce BCA Protein assay, gel electrophoresis and protein transfer to a specialized membrane, and protein staining procedures. The poster present for this project shows my progress up to date. The impact of this research is to help set the protocols needed for the BIOL 431 laboratory research course.

Goals of URES 197My goal for URES 197 was to troubleshoot the protocols associated

with observing protein expression and establish a set of working protocols for the BIOL430 class to use next fall.

Methods and Materials The first protocol for the experiment was to isolate total protein from

yeast and measure the concentration to determine the correct amount of sample to load on the western blot. This was done by first making a 24 hour 3mL broth culture of yeast. Using the FastPROTEIN Red kit and FastPrep Instrument, protein was isolated.

Following protein isolation, the concentration using the Pierce BCA Protein assay kit. I made 9 standard concentrations in order to create a standard curve graph. This way, I could determine my sample concentration from the graph. I micropipetted each standard sample and unknown sample into individual wells of a 96 well plate and measured their absorbance at 560 nm using the spectrophotometer. I saved each value and made a standard curve graph using Microsoft Excel and I determined the unknown concentration of Cin8p using this graph.

Conclusions Although detection of my yeast samples were successful in both the

Coomassie Blue and Immuno-Blot Assay, the Cin8p detection was not specific in the Immuno-Blot Assay. This could be due to the fact that not enough Tap-Tag antibody was added to the first wash during Immuno-Blot, thus not allowing enough attachment of antibody to target protein to allow a proper detection. Other errors could be due to mechanical and personal error. Another student this summer will continue to troubleshoot this experiment this summer to fix this problem.

Although this experiment did not yield the desired results, I made a great improvement from last fall during BIOL 430. I was able to successfully detect proteins from my sample, as well as master techniques that will be helpful for my future in the molecular biology field.

Establishing Protocols for Measuring CIN8 Expression during the Saccharomyces cerevisiae Cell Cycle

Elizabeth Doswell and Deborah Zies — Department of Biology, University of Mary Washington

ResultsFrom my protein isolation and concentration experiment, I

determined that the mean protein concentration from my three samples 1.367µg/µL. I determined the average of each standard absorbance and made a standard curve using Microsoft Excel. Comparing my sample absorbance to the standards I determined I had enough protein to run the rest of my experiments.

The Coomassie Stain was positive and successfully demonstrated a protein ladder and some total protein, but there are fewer protein bands than expected.

The Immuno-Blot Assay was positive in that a ladder appeared after a couple of hours, but the ladder that formed was identical to the one that formed in the Coomassie experiment. This indicated that this was not specific to only Cin8p expression in the sample.

Fig . 2 . Pho tograph o f b lank membrane following western blot and immuno-blot of S. cerevisiae with wild type CIN8

Following the protein concentration procedure, I ran a Western blot of the yeast total in order to visualize total protein and Cin8p expression in S. cerevisiae. Using a 12% Tris gel, I loaded wells 1, 3, 7 and 9 of the gel with a standard and loaded wells 2, 4, 8 and 10 with my yeast sample. I ran my gel for about 40 minutes (or until the samples reached the bottom of the gel) at 200 mV. Following the gel electrophoresis, I transferred the gel to a membrane. This involved placing the membrane against the gel and enclosing the two between two sheets of filter paper. These compontents were placed in the same container as the gel electrophoresis. I ran the Western blot transfer for 30 minutes at 100mV. Once the transfer was complete, I dismembered the cassette and discarded the gel. I washed the membrane in methanol and then cut it into four sections, so I could perform various experiments. The membrane pieces were stored in filter paper at 4°C.

Fig. 1 . Hoechst s ta in of S. cerevisiae cells with CIN8 deletion. Cells demonstrate a binucleation.

For color detection using Immuno-Blot Assay, I obtained another membrane sample from the freezer and washed it in a mixture of TBS solution, powdered milk and DI water for 30 minutes. This was to ensure the membrane was clean. Following which, the membrane was washed in TTBS solution, Cin8p Tap-Tag antibody and powdered milk for 12 hours. I removed the membrane and washed it in TTBS for 10 minutes then soaked it in a solution of TTBS, standard antibody and powdered milk for 2 hours. After this time I washed the membrane in TTBS for 10 minutes and began color detection. I submerged the membrane in a solution that contained color reagents A and B and waited for results. Because I did not see immediate results I left the membrane in the color staining for a couple of hours.

IntroductionIn the fall semester, I took BIOL 430 – Molecular Biology of the

Gene, a research intensive class that focused on the principles and technique it takes in the molecular study of genes. Throughout the course of the semester, my research group designed a project that dealt with observing the phenotypes of the mutant and wild type gene CIN8, formally known as Chromosome Instability gene.

In S. cerevisiae, CIN8 is a kinesin gene, utilizes ATP for energy, whose main purpose is microtubule formation and function during anaphase mitosis. Prior to anaphase initiation, CIN8 works to ensure proper spindle fiber formation, specifically on kinetochore clusters in order to allow proper attachment of spindle fibers to the centromere of the chromosomes. As well, CIN8 works during anaphase to monitor proper spindle elongation, shortening, and speed at which they drag chromosomes to opposite cell poles. However in a deletion of CIN8, proper spindle formation and function would not be possible, leading to the improper chromosome separation and thus improper cell division.

For our class experiment, we hypothesized that in the wild type yeast would have the highest level of CIN8 production at anaphase and would have normal spindle formation and normal cell division. Additionally, we hypothesized that in yeast that contained a deletion of CIN8, there would be improper spindle formation and failed cell division that we could observe as binucleated cells.

We were able to observed the phenotype of mutant (knock out) CIN8 (figure 1). We were, however unable to observe the wild type expression of CIN8.

Fig. 1. FastPep machine demonstration (right)

Fig. 2. 96 well plate with standards and unknowns (left)

In order to visualize protein expression, I used two color detection procedures: Coomassie Blue stain that should show total protein and the and Immuno-Blot Assay technique that should detect only CIN8.

For Coomassie Blue stain, I obtained one membrane sample from the freezer and submerged it in Coomassie Blue stain for 10 minutes. Following the stain, I washed the membrane in an Acetic Acid, Ethanol and water mixture for 10 minutes to halt the staining process and decolorize the membrane. Following this I repeated the was until individual protein bands could be observed.

Fig. 1. Diagram of Western blot procedure (left)

Fig. 2. Western blot gel run (right)

Fig. 3. Western blot membrane transfer (left)Fig. 4. membrane transfer set up (right)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 500 1000 1500 2000 2500

Abs

orba

nce

(nm

)

Protein Concentrations (µg)

Fig. 1. Standard curve generated to determine unknown protein concentration

Fig. 2. Coomassie Blue Stain membraneFig. 3. Immuno-Blot Assay membrane