BIOL 2025 Lab #10 Name_______________
Proteomics: Western Blot
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The Western Blot
Western blotting is an immunodetection technique used by proteomic scientists to detect and
quantify specific proteins in complex biological samples. First, proteins are extracted from a
sample of cells or tissue. Extracted proteins are loaded into a sieving gel matrix and separated
according to size using an electric current, that is, by electrophoresis. Proteins separated by
electrophoresis are then transferred or “blotted” from the gel onto a paper-like membrane. A
specific antibody, engineered to bind only to the protein of interest, is added to the membrane.
This antibody is attached to a compound that causes a colored reaction, enabling scientists to
detect and quantify a single protein of interest from hundreds of other proteins in a sample with
high accuracy.
Western blotting can categorically identify a specific protein among hundreds or thousands of
other proteins within biological samples. This surefire method of identifying proteins is based on
two distinguishing features of proteins: molecular mass and antibody binding specificity.
Bioscience researchers use western blotting to identify proteins, quantify protein expression
levels, and determine whether proteins have undergone posttranslational modification. Because
it is so accurate, western blotting is the method of choice used to confirm positive test results for
HIV, lupus, or bovine spongiform encephalopathy (BSE; mad cow disease).
In this lab, protein gel electrophoresis and western blotting will be used to specifically identify a
subunit of a myosin light chain from the many thousands of proteins comprising the muscle
tissues of different fish. Myosin light chain proteins will be compared from different species for
variation, commonality, or evolutionary divergence. Are there discernible differences between
the myosin proteins extracted from the species you are investigating? What are they? How
might these variations occur, and why? How might variations in myosin between species be
used to determine their evolutionary relationships?
Muscle Proteins and the Myosin Light Chain
The basic functional units of animal muscle, myofibrils, are bundled to form muscle fibers.
Each myofibril consists of a linear series of contractile units called sarcomeres (see Figure
17.42 below). Sarcomeres are precisely arranged assemblies of actin and myosin protein
filaments. Up to fifty percent of skeletal muscle is comprised of myosin protein. Thin actin
filaments are aligned with thick filaments of myosin in a parallel and partly overlapping manner.
Myosin has a 3-D structure composed of six subunits: two myosin heavy chains with molecular
masses of 200 kiloDaltons (kD) and four myosin light chains with molecular masses ranging
from 15 to 25 kD. The heavy chains have a long tail, a neck, and a globular head region. The
two heavy chain tails wind around each other and in turn encircle the tails of neighboring myosin
molecules, weaving long cable-like structures that form tough myosin filaments (see Figure 17-
44 below). The head regions protruding from the cable filaments interact with thin actin
filaments. Two myosin light chain proteins wrap around the neck of each myosin globular head
region and help to regulate the contraction of the myosin protein.
