Bio15 Lab3

Bio15 Lab3 - Sutaria 1 Lab 3 Qualitative Analysis of...

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Sutaria 1 Lab 3: Qualitative Analysis of Protein Purification Ravi Sutaria HB 3390 Lab Partner Erika Graham TA: Melissa Lathrop Lab Section 7
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Sutaria 2 I. Introduction The objective of the three protein labs was first to separate a mixture of proteins by gel filtration chromatography, then to quantify the amount of protein in each fraction using a technique called the Bradford Assay (colorimetric assay), and finally to run an SDS (Sodium Dodecyl Sulfate) gel electrophoresis to provide a way to asses the success with which the protein mixtures were fractionated and to determine the molecular weight (size) of the proteins in each fraction (Biology 15 Lab Manual , Page 1). In gel electrophoresis, an electrical field is applied across the gel to mobilize the proteins and cause all the molecules to migrated through the gel. The mobility of the protein molecule is directly proportional to its net electrical charge and inversely related to size (However, treating with SDS gives the proteins a net negative charge, resulting in a separation of proteins based solely on size) (Biology 15 Lab Manual, Page 1). The electrophoresis gel s a polymerized matrix of gel that allows migration of proteins that in our case were “denatured” into unfolded polypeptide chains by the SDS (Biology 15 Lab Manual, Page 2). Figure 1 – SDS Gel Electrophoresis) [Wikepedia, Page 1]
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Sutaria 3 II. Methods A. SDS Gel Electrophoresis We began the experiment by numbering 1.5 ml microtubes with the fraction number of the protein samples selected (protein samples 1, 3, 6, 9, 14, 16, 18, 20, 23, 24, 25, 27, 29) as well as a Protein Mixture (PM) 1:10 provided by the Lab instructor. Each microtube was loaded with 5 microliters of 4X PAGE sample buffer (composed of 8% SDS, 40% Glycerol, 20% 2-mercaptoethanol, and a pinch of Bromophenol blue dye in .250 M Tris – HCL buffer, pH 6.8) and 15 microliters of the chosen column fractions and “PM 1:10” for a sum total of 20 microliters in each microtube (Biology 15 Lab Manual, Page 3). These tubes were then immersed in boiling water for 3 minutes to denature protein, allowed to cool, and then centrifuged for a homologous mixture. A 15 % SDS PAGE Gel Sandwich was used that had a 15 tooth comb that served as the template for the wells. In the wells first 10 microliters of the protein molecular weight standard was loaded in well 1, followed by 20 microliters of all the chosen protein fractions, and also 20 microliters of the “PM 1:10” (Biology 15 Lab Manual, Page 4). After the wells were loaded, we began running the gel at a voltage of 180 volts (current of 50 mA (since two gels were run [25mA each]). After 5 minutes of running, the voltage was raised to 200 volts and kept constant to speed up process. In about an hour the blue tracker dye was approximately 5mm from the bottom of the gel and the gel was removed (Biology 15 Lab
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This note was uploaded on 04/25/2008 for the course BIO 015 taught by Professor Rotating during the Winter '07 term at Dartmouth.

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Bio15 Lab3 - Sutaria 1 Lab 3 Qualitative Analysis of...

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