BE115+-+Lab05 - Lab 5: Protein Quantification 1...

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Page 1 of 7 Bioengineering 115: Cell Biology for Engineers Laboratory 5: Protein Quantification Last updated: January 21, 2011 Lab 5: Protein Quantification 1 Introduction Calculating the amount of protein in a particular sample is the first step for most protein analysis methods. In this class, we’ll be doing protein gel electrophoresis, so let’s use that as an example. If protein concentration is unknown, then insufficient protein may be loaded, which will prevent identification of the components of interest. Alternately, too much protein will distort the separation pattern, sometimes to the extent that the high resolving power of the method is compromised. To avoid these problems, at least an estimate of the protein concentration to be loaded onto an electrophoresis gel is required. There are several spectrophotometric assays for protein concentration that differ in sensitivity, specificity, cost, and convenience. The following experiment will introduce two of the most commonly used methods: the Coomassie (or Bradford) assay and the bicinchoninic acid (BCA) method. Notes: For these experiments you and your partner will perform all steps together. Where large equipment is required, you will be allotted a set amount of time at that piece of equipment. More details are listed in the protocol section below. This lab contains a lot of different tubes, cuvettes, and wells. Take care to label everything. As with any protein estimation, the standard protein chosen for the estimation should be of a similar type to that which is being assayed. In general, use bovine serum albumin (BSA) for globular protein estimation and IgG for glycoproteins. We’ll be using BSA for our standard in this lab. Keep in mind that your cuvette or 96-well plate must be kept clean of marks, fingerprints, or scratches. If any of these interrupt the light path, your readings will reflect not only the absorbance of the liquid inside, but also the interference from the dirty cuvette.
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Page 2 of 7 Bioengineering 115: Cell Biology for Engineers Laboratory 5: Protein Quantification Last updated: January 21, 2011 2 Objectives To prepare dilutions of a protein standard and dilutions of your protein lysates.
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BE115+-+Lab05 - Lab 5: Protein Quantification 1...

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