Creatine Kinase

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Protein Purity and Activity

• Creatine Kinase
• as a Marker of
• Muscle Damage

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Purpose

• Introduction to laboratory workspace
• Hands-on experience with lab equipment
• Learning the meaning of research
• GraphPad Prism 4 intro.

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Goal

• Our goal in this activity was to find the amount
  of Creatine Kinase in four different samples of
  total cellular proteins. When that was found, we
  also wanted to find out how much protein was
  present in each sample.

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Creatine Kinase

• Type of protein which is found in muscles.
• looked for when suspected that muscles are torn
  or damaged, such as Heart attacks.
• Not normally found in blood stream unless there
  has been damage.

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Protein purification

• Use solutions to separate the different types
  of proteins.
• Sticks and separates by using electric charges.
• Column chromatography -drips solution through
  to separate the proteins.

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Gel Electrophoresis

• Proteins are separated by the sizes as they
  flow through the gel.
• Smaller proteins at the bottom and the larger
  at the top.
• Coomassie Blue- makes the proteins visible.

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2nd Chemical used to detect Creatine

Creatine a-napthol Diacetyl ? Colored
Complex (pink)
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THE TEST TUBES
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Spectrophotometer?
In order to find out exactly how much creatine
kinase was in our test tubes, we used a
spectrophotometer (measures the wavelength that
is absorbed by a given sample.)
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Protein Assay

• In our research we were trying to find out
  which one of our samples held the most protein.
• To measure the protein in our samples we used a
  spectrophometer.
• We added Bca ( bichinichoninic acid ) to form a
  blue complex in the presence of proteins.
• We found that high salt had the most protein.

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We measured each known substances three
times. High salt contained the most protein.
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We measured eight unknown substances. Some of
the unknown substances were negative.
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Summary / Result
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These graphs show the nmol creatine/ul in the
samples extract, flow thru, low salt, and high
salt. The graph on the left shows Jason and
Aarons results. The graph on the right shows
the whole groups average. We lost our creatine
activity throughout the experiment. The group
all together found creatine activity in all of
the samples.
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These graphs show the ug Protein/ul through the
samples Extract, Flow Thru, Low Salt, and High
Salt, In group ones experiment the Protein was
almost spread evenly throughout the samples. The
whole groups Protein was low on the flow through
and increases as it goes through the Low Salt and
High Salt.
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During our studies of protein and creatine
kinase, Dr. Graber informed us of useful
information. The work we had accomplished was
tough, but we had an excellent time. What we
really learned was that it isnt search- its
RE-SEARCH