Synapsin Staining Report

The null hypothesis will be that the test tubes with an increase in temperature, pH values, enzyme concentrations, and substrate concentration will have a very small color change or no color change at all. The alternate hypothesis is that the test tubes containing an increase in temperature, pH values, enzyme concentrations, and substrate concentration will all have an intense color change; the more the change, the more intense the color change will be.

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

1) Apply the stain to your first unknown slide and examine it under the microscope.

Tetrahymena in the 5% ink showed a higher stained vacuole count that those in 1% ink. This supported our hypothesis that the Tetahymena in a higher concentration of ink would show more staining, because there is more ink around for them to consume. Differing results were found in a similar experiment by Bazzone (2000), as cells in a higher ink concentration showed fewer vacuoles than those at a lower ink concentration. This experiment followed the same procedure, but expanded the ink concentrations to include 10% ink and examined cells at the timepoints 2, 10, 20, and 30 minutes (Bazzone, 2000). This study did not find an explanation for why these results occurred (Bazzone, 2000). Since our numbers of vacuoles of 1% and 5% ink were nearly identical for each time period, it is possible that if we repeated the experiment and counted more

Then we carefully submerged the nitrocellulose sheet on the transfer buffer for about 5 minutes, and set up the transfer apparatus in the method shown in the lab outline week 2 step 3-6. Afterwards we took apart the transfer apparatus and detached the nitrocellulose sheet. Then we put the nitrocellulose sheet for 5 minutes into the blocking buffer, and we left nitrocellulose sheet in a container till week 3. The second part of week 2 was staining half our PAGE gel from week 1. Which consisted of pouring deionized water and use the shaker machine for 5 minute intervals, and repeating the the process three times. Then the Fresh Coomassie stain was added to the gel and was rinsed (UFV BIO 202 Lab,

The drops of crystal violets, approximately 15 drops, were flooded until the smear were all covered and then allowing resting for one to two minutes. After two minutes, the slide was titled over the sink and washed off, with the distilled water bottle, by aiming the stream of water above the smear. The specimen appeared blue-violet when observed with the naked eye. The drops of Gram’s iodine were applied on the slide until covered and then allowed to react for one minute or more. After the time elapsed, the slide was rinsed again with distilled water following immediate drops of Gram stain decolorizer added one drop at a time.

Group 1, the gel picture and Andersons experiment are closely related. The binding did occur, in the 1st lane of group 1the dye was free therefore no binding occurred because no Bovine Serum Albumin (BSA) was present in the mixture. While in lanes 2 to 4 binding did occur because there was BSA in the mixture, with the higest binding occurring in lane 4, this is because there was no buffer to interrupt the binding. (saturation).

The control used in the experiment was water. For each trial we set up, it was to see the results between our assigned extract and water. This was useful in interpreting our results because our results were based on how the insects responded. If they had no preference to either strip, attraction to the plant extract, or if they

The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique used to measure the concentration of an analyte (usually antibodies or antigens) in solution. In the practical anti-BSA antibodies that had undergone serial dilutions were added to a BSA solution in an ELISA plate with goal of seeing how the concentration of anti-BSA antibodies would affect the colour change of the BSA solution. The results clearly showed a direct correlation as the more diluted the anti-BSA antibody solutions became the lower the Wavelength readings at 405nm, which showed that there was less of a colour change.

a)What type of stain would you use to determine whether there had been any changes in total cell number following administration of the neurotoxin?

On a thin chromatography plate, five spots were placed ( as shown in table 2) and the plate was developed using chloroform/methanol. This was later visualized with dragendorff’s reagent under the UV light. All separated components were observed, identified and recorded.

Reticulitermes termites can be found in most southeastern regions because of the warm and humid conditions. The termites in this experiment are housed inside a petri dish that can be described as pale insects three to twenty millimeters long in body length, and can also be commonly known as “white ants”. Termites live in organized colonies and are defined by divisions of labor. Termites are also considered to be social insects inside their colonies where they work together to bring food and water back to their colonies for survival. There are different levels in a termite colony, which consist of workers, soldiers, and reproductive (Watson, 1985).

A clean toothpick is used for every standard to place a small spot of the dye on the cross mark of the paper in the appropriate column. The spot cannot get too large, or the chromatogram may be streaky and difficult to read. Spots should not be greater than 2 mm in width. The spot may have to dry and more sample will have to be added to get a dark spot without the spot becoming too diffuse.

After wearing the gloves we obtained a chromatography vial from professor and label it with my and my peer initials. We dried up the chromatography vial in fume hood and added 1 ml of chromatography solvent to the vial. Then we took a chromatography strip and measure it 1.5 cm with ruler from one end of the strip and drew a line with pencil we cut two small pieces below the pencil line to form a pointed end. We applied spinach on the strip using quarter to rub the spinach leaf on the line that we drew on the strip and put it into the chromatography vial and placed that in fume hood. We observed as the solvent was moving up the chromatography strip by capillary action. When the solvent was reached approximately 1 cm from the top of the strip then we removed the cap from the vial. We took out the strip from the vial using forceps and marked up the location of the solvent front because it evaporates quickly. We measure out the distance as well as the pigment in order to find out the rf value. Moreover we compared rf values to the one in reference list in order to identify the

A drop of blood is added into each test tube and they are left for 5 min.;