Gel Electrophoresis

Abstract

The main objectives of this experiment were to prepare a polyacrylamide gel and to use the technique of gel electrophoresis to separate, analyze and quantify the isoenzymes of lactate dehydrogenase. The five isoenzymes of lactate dehydrogenase were separated based on their relative charges, which is proportional to the distance travelled through the gel. The five isoenzymes of lactate dehydrogenase can be ranked in order of increased distance travelled in the polyacrylamide gel from, M4 < H1M3 < H2M2 < H3M1 < H4. The absolute quantity of LDH isoenzyme H4 was determined to be 8.73 g through the use of the VGAU-Vakili3000. This value was obtained by comparing the relative areas underneath the plots of optical density measurements for the standard H4 with concentration of 4.8 g/15 L and the purified LDH from Experiment 2. The total percentage of protein in fraction 4 that was LDH was found to be 0.0638%. This extremely small percentage suggests a loss of LDH isoenzymes in the experiment, which can be attributed to experimental errors, including denaturation of the isoenzymes and loss of the isoenzymes via the buffer during electrophoresis.

Introduction

The main goal of this experiment was to prepare a polyacrylamide gel and subject it to polyacrylamide gel electrophoresis (PAGE) to analyze and quantify the different isoenzymes of lactate dehydrogenase1. The electrophoresis was run on a crude extract and a purified fraction of lactate dehydrogenase obtained from Experiment 2, along with a standard isoenzyme. The quantity of each isoenzyme was determined through the use of an instrument measuring the optical density of the gel1. The absolute quantities of isoenzymes in the gel were compared to the total protein concentration of the purified fraction from Experiment 2, to determine the success of the purification of lactate dehydrogenase isolation.

Lactate dehydrogenase (LDH) is an enzyme that catalyzes the...