Sea Urchin VE Removal

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Sea Urchin VE RemovalPrediction of Molecular
Weights of unknown

• ByMichael Dinse
• Elizabeth Gutierrez
• Maria Uribe

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Purpose

• Observe Vitelline envelope peptides that have
  been isolated by two chemical methods
  (alpha-amylase DTT) and a mechanical isolation
  (manual) and predict their molecular weights
  through a process of analysis, measurments, and
  various calculations.

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Method/Process

• Data Collection
• Measure four different gels, each containing
  seven different bands (1 standard, 2
  alpha-amylase, 2 DTT, and 2 manuals)
• Model the standards using the following methods
• linear, linear (Using Log MW), quadratic, cubic,
  special cubic, and special log.
• Determine which one is the best model in order to
  predict our unknown data set.

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Tools

• Measurements of Gel bands were made using images
  from Photoshop.
• Calculation and Analysis of the data was
  completed using Excel and Minitab.

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Description of the Vitelline Envelope, AA, and DTT

• Before we proceed it is important to give a
  brief description of the Vitelline Envelope and
  the two chemical methods (alpha-amylase DTT).

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The Vitelline Envelope

• Composed essentially of proteins, the vitelline
  envelope acts as the protective layer just above
  the eggs inner membrane.
• In sea urchins this protective layer is in the
  eggs jelly.

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Fertilization of a Sea Urchin
http//www.mhhe.com/biosci/cellmicro/kalthoff/samp
le_ch04.pdf
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Alpha-amylase

• Alpha-amylase (1,4-alpha-glucan
  4-glucanahydrolase Ec 3.2.1.1) are ubiquitous
  enzymes which catalyze the breakdown of amylose
  and amylopectin through the hydrolysis of
  internal alpha-1,4-glycosidic linkages with net
  retention of anomeric configuration.
• http//www.yorvic.york.ac.uk/projects/2/2.2.3.htm

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Alpha-Amylase

• Alpha-amylase are found in a diverse array of
  industrial processes..including..the
  pharmaceutical industry.
• http//www.yorvic.york.ac.uk/projects/2/2.2.3.htm

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DTT (Dithiothreitol)

• Dithiothreitol (DTT) is commonly used in
  biochemical research to protect sulfhydryl groups
  from oxidation or reduce disulfide linkages to
  free sulfhydryl groups in proteins and enzymes.
• http//www.wcaslab.com/tech/Dithiothreitol.htm

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Data Collection

• Measured four different gels using Photoshop
• Gel 1 12 Method Comparison
• Gel 2 replicate of Gel 1
• Gel 3 10 Method Comparison
• Gel 4 replicate of Gel 3

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Data Collection

• Each gel that was measured contained seven
  different bands
• 1 Standard
• 2 Alpha-Amylase (AA)
• 2 Dithiothreitol (DTT)
• 2 Manual
• Each band was measured three times in order to
  obtain a more accurate reading.

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Models Analyzed

• Using the standard bands the following models
  were analyzed
• linear/linear(Using Log MW).ymxb
• quadratic.....yabxcx2
• cubic...yabxcx2dx3
• special cubic...yabxcx3
• special log. yabxclnx

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Model/Analysis

• For each of the five different models, predicted
  values, standard deviations, and the R-squared
  was calculated.

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Best Model

• After analyzing the r-squared and the difference
  between the confidence intervals, the best fit
  model was chosen.

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Best Model

• These are our results for the best model.
• Gel 1 12...CUBIC
• Gel 2 replicate of Gel 1.CUBIC
• Gel 3 10.SPECIAL CUBIC
• Gel 4 replicate of Gel 3...SPECIAL CUBIC

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Why did the Cubic Model best predict the data set
for Gel 1 and Gel 2?

• GEL 1
• GEL 2

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Why did the Special Cubic Model best predict the
data set for Gel 3 and Gel 4?

• GEL 3
• GEL 4

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Analysis of unknowns

• Once a best model was determined predicted values
  for the six lanes of unknowns were computed using
  the equations for the best fit models.
• In order to convert these values to molecular
  weights the antilog of the predicted values was
  taken.

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Analysis Continued...

• Once Molecular weights were known averages of the
  two lanes of AA, DTT, and manual were taken.
• Using the manual column as our reference the
  averages for AA and DTT were compared.

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Molecular Weight (Band) Comparison
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Molecular Weight (Band) Comparison Continued...
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Conclusions

• From the analysis it can be demonstrated that in
  the case of the 12 gels the method which gave
  results similar to those which were obtained
  manually was that of AA.
• The reasoning behind this conclusion is due to
  the fact that extra bands were obtained in DTT
  which did not exist in the manual obtained
  unknowns.

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Conclusions

• From the analysis it can be demonstrated that in
  the case of the 10 gels the method which gave
  results similar to those which were obtained
  manually was that of DTT.
• The reasoning behind this conclusion is due to
  the fact that DTT obtained more bands in common
  with the manual than AA.

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Conclusions

• In the 10 gel, one standard band was lost.
• In the 10 gel, each lane lost the lower band
  (which went to the die front).
• In the 10 gel, the bands were not as dense as in
  the 12 gel.