Question

PARTB Experiment 1 (201). Molecular Weight Determination The 1-1. You will be in the wild with me is found in rabbit serum. This protein is called serumbumin. The sandard unknown proteins and the rabbit serum prins for this experimentarehe pre- tined to allow you to follow the portion during crophews Background Information A first step in the analysis of a protein in the molecular biology laboratory frequently involves determination of its sie or molecular weight. The molecular weight of a protein may be determined using a variety of techniques. Gel electro phoresis in the presence of SDS is one of the most common methods since it is relatively simple to perform and does not require elaborate equipment. When an SDS denatured protein is separated cloctrophoretically, its rate of migration from the point of application toward the positively charged electrode is proportional to the log 10 of its molecular weight. When the molecular weights of several standard proteins are known, their mobilities can be ploted as a function of molecular weight to give a linear calibration curve (see Figure 1-1). The molecular weight of an unknown protein can then be estimated from this calibration curve. In practice, protein standards and unknowns are electrophoresed on adjacent lanes of the same gel. Following electrophoresis, the relative migration of standards and unknowns are determined and the molecular weight of unknowns calculated. Table 1-1. Molecular Weights of Standard Proteins. Standard Protein Molecular Weight Diagram of Gel (dos) Sample Well 1. Bovine Serum Albumin Dimer 132,000 - - 2. Bovine Serum Albumin Monomer 66,000 3. Ovalbumin 43.000 4. Lysozyme 14400 Le Bromophenol Blue *The terms "molecular weight and dalton" are used interchangeally in this manual. For example, a 20.000-dalion peotel has a molecular weight of 20.000 A dalton is a unit equal to 1.0000 on the shomic mass scale this unit is very nearly equal to that of a hydrogen atom. The average amino acid resuela a protein is 120 daltons. Thus, a protein with a molecular weight of 20,000 contains 167 amino acid residues (20,000/120 - 167). Figure 1-1. Typical Calibration Curve of Standard Proteins. 200,000 Objective To determine the molecular weight of three proteins Materials 100,000 000- MOLECULAR WEIGHT (LOG SCALE) 1. The solutions and materials required for electrophoresis and sample handling (see the Instructor Guide). II. The samples listed below are provided for this experiment. 1. Pre-stained protein standards: The molecular weights of these proteins are given in Table 1-1. These proteins are stained blue 20,000 2. 50 Pre-stined unknown protein: A protein sample of unknown molecula weight. The protein is stained blue. Pre-stained unknown protein It: A protein sample of unknown molecu weight. The protein is stained blue 20 3. 10 DISTANCE MIGRATED (mm) 491 L ab lest In this exercise, you will determine the molecular weights of two unknown proteins by comparing their electrophoretic migration with the migration of standard proteins. The molecular weights of these standards are given in Table 1 1994 by John N. Andenen

Answer 1

The Y axis is molecular weight (0-140 kDa), while the X axis is distance traveled by the protein in mm.

Y Axis Title 23 - 28 33 38 43 X Axis Title 48 3 58

The red dots are standard proteins and Rabbit serum protein.

Molecular weight of unknown protein 1 (with 26 mm distance traveled) is 103,377 Da.

Molecular weight of unknown protein 2 (with 58 mm distance traveled) is 15,865 Da.

The unknown proteins 1 & 2 are shown in blue color.