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LS1a Fall 2014
Lab 3 In-Lab Activity
The following activities are to be completed during lab, while your gels are running.
Due date: This in-lab assignment is due one week after completion of the lab at the start of your
next section, along with the post-lab assignment.
1. (10 points) Polyacrylamide gel electrophoresis separates molecules based on their molar
mass.
a) (5 points) We measure protein mass in units of “Daltons” (“Da”), named after John
Dalton who helped establish our understanding of the atomic nature of matter. One
dalton (Da) equals one atomic mass unit, such that one carbon equals 12 Da and one
nitrogen equals 14 Da (please refer to the periodic table at the end for atomic mass
units). Calculate the molar mass in Da for tryptophan:
b) (5 points) The molar mass of a protein is determined by its length and amino acid
composition. Calculate the molar mass of GFP by looking up the protein’s amino acid
sequence and entering its sequence into a program called Protparam (i.e., “Protein
parameter”).
i) First go to Pubmed (www.pubmed.com), and search for the amino acid sequence of
GFP by typing “green fluorescent protein aequorea victoria” into the search field
and choosing “Protein” from the scroll menu to the left of the search field
ii) Select the first result, and click on “FASTA” to get the amino acid sequence of the
protein:
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iii) Select and copy the amino acid sequence:
iv) Open up a new tab in your web browser and go to http://web.expasy.org/protparam/,
and paste the sequence of the protein into the search field that says “Or you can paste
your own sequence in the box below.” Click on “Compute parameters.”
v) Record the “molecular weight” of GFP. This is the molar mass that will determine how
far your GFP protein will migrate through the polyacrylamide gel.
Molecular weight of GFP:
Da (or 26.9 kDa)
2. (10 points) Once we separate our proteins using the polyacrylamide gel, we detect where
our proteins are by staining the gel with a dye called Coomassie blue, shown below:
Coomassie blue primarily interacts with positively-charged amino acids. Given the pKa
values of Coomassie Blue’s (-SO3H) groups, what is the optimal pH to apply the Coomassie
dye to our gels in order for it to interact with the positively-charged side chains of histidine,
lysine, and arginine?
3. (10 points) How does SDS ensure that a protein migrates through the gel according to its
molecular mass instead of according to its net charge or its shape?
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4. (10 points) Shown below are two SDS-PAGE gels after they have been run and stained with
Coomassie Blue. Each gel contains a ladder with the sizes of its bands indicated in
kilodaltons (kDa).
a) (5 points) For each gel, identify each bands that are not part of the ladder and provide
an estimate of their mass in kDa. Indicate which protein in each (non-ladder) lane is in
the highest concentration.
b) (3 points) While tryptophan is the largest amino acid, an average amino acid has a molar
mass of about 110 Da. Using this value, estimate the number of amino acids in the most
concentrated band in lane 1 for the gel on the left.
c) (2 points) Shown below (next page) are the results of an experiment in which a pair of
proteins with masses of 19 kDa and 87 kDa is treated with different concentrations of
protease. Each lane represents an experiment in which the concentration of the 19 kDa
and 87 kDa proteins is constant, but a different concentration of protease was added.
Label the sizes of the protein bands in each lane. Which lane contained the highest
concentration of protease? Briefly explain how you can tell.
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Atomic mass units
(“Da,” or “daltons”)