University of California, Riverside BCH 162 _Experiment 4: Purification of recombinant dLDH from E. coli by metal-ion affinity chromatography

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University of California, Riverside BCH 162  Experiment 4: Purification of recombinant dLDH from E. coli by metal-ion affinity chromatography   Lab Dates: 2/1-2/15 Lab Partner: Lab Report... Due Date: 2/22/18   Objectives Purification The goal of purification is to isolate the specific enzyme lactate dehydrogenase in chicken breast muscle through tissue homogenization, ammonium sulfate precipitation, and pseudo affinity chromatography. Bradford Assay The purpose of Bradford Assay is to help determine the amount of concentration of protein in each of the fractions taken from the experiment. Procedures Homogenization in presence of PMSF and 2-ME The homogenization process starts with the addition of PMSF and 2-ME to chicken tissue which will then be mixed in a blender. The homogenate will be transferred to centrifuge tubes, centrifuged for 20 minutes, and filtered through cheesecloth to collect the supernatant. AmSO4 Precipitation Ammonium sulfate will be slowly added to the supernatant at a ratio of 0.24g for each 1ml of supernatant while mixing the two in a beaker while in the cold room. This solution will be transferred to tubes and centrifuged for 20 minutes giving the 40% cut. Ammonium sulfate will be added again to the 40% cut at a ratio of 0.13g for each 1 ml   of 40% cut, mixed and transferred to the centrifuged for 20 minutes which will give the 60% cut. Resuspension and Desalting The ammonium sulfate pellet will be resuspended with TRIS-ME buffer and kept on ice before transferred to a Sephadex G-25 column. After transferring the resuspended sample to the column, the sample will be desalted with TRIS-ME buffer and collected. Cibacron Blue Chromatography The desalted sample will be loaded into the Sephadex G-25 column and first washed with TRIS-ME buffer and the elution will be collected and have an absorbance reading. The elution will go through a second wash which contains NAD+ and the elution will be collected and have an absorbance reading. The elution will go through a third wash with TRIS-ME buffer and the elution will be collected and have an absorbance reading. The elution will go through the last wash with NADH buffer and have fractions collected along with absorbance readings. [Show More]

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