Month: December 2018

Statistical analysis

Statistics are one of the most important tools for a scientist. They allow scientists to test hypotheses. Perhaps you would like to  know if a treatment makes your favorite organism grow better. Statistical analysis would let you determine the probability that the treatment increases growth. The below video shows you how to perform statistical analysis (t-test) in Excel.

Choice of t-test (paired vs two sample equal variance vs two sample unequal variance)

Ideally statistical analysis should be planned before the experiment is setup. Paired t-test has more statistical power than the other two types of t-test because it helps minimize the effect of nuisance factors that confound the experiment results. An example of how to setup an experiment to take advantage of paired t-test in plants is as follows. Control and treatment for each replicate would be planted side by side so that the replicate was in a block. Doing this minimizes the effect that different growing positions have on each replicate. A paired t-test takes this into consideration and assumes while the absolute measurement values might change for different replicates, the mean difference between control and treatment for each replicate should be conserved. The best part of using a paired t-test or other blocking strategy, is that replicates do not need to be completed at the same time. This way there is no real limit to the number of replicates for an experiment.

Quantifying bands on SDS-PAGE using ImageJ

The below video shows you how to use ImageJ to quantify bands on a SDS-PAGE gel image. In this example, bands are normalized to the total protein in the lane.

Here you can download the sample gel image from the video to practice with. Note that many commercial gel docs, like Bio-rad, come with software for quantifying SDS-PAGE bands. In contrast, ImageJ is a free alternative that can work with any gel images including ones generated with an office scanner. Almost the same procedure in the video can be used for quantifying nucleic acid gels or even features on non-gel images.

Enzyme kinetics is the study of enzyme catalyzed chemical reactions. Enzymes speed up reactions by binding to the transition state of the reaction. A detailed overview on enzyme kinetics can be found here. Increasing substrate concentration speeds up a reaction to a certain point, Vmax. Vmax is the maximum reaction rate that can occur and adding more substrate will not increase the rate of the reaction because the enzyme binding site has become saturated. The Michaelis–Menten constant, Km, is defined as the substrate concentration in which the reaction will go at half maximal rate. In order to determine the Km and Vmax of a reaction you will first have to calculate the reaction rate at several different substrate concentrations.

Determining reaction rates

This video shows you how to calculate reaction rates in Excel for urease, an enzyme that catalyzes the hydrolysis of urea to carbon dioxide and ammonia.

Determining Km and Vmax

Km and Vmax can be determined once reactions rates at different substrate concentrations have been calculated. This video shows you how to calculate Km and Vmax in Excel.