1. Sea Urchin VE Removal…Prediction of Molecular Weights of unknown By:Michael Dinse Elizabeth Gutierrez Maria Uribe

2. 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.

3. 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.

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

5. 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).

6. The Vitelline Envelope Composed essentially of proteins, the vitelline envelope acts as the protective layer just above the egg’s inner membrane. In sea urchins this protective layer is in the egg’s jelly.

7. Fertilization of a Sea Urchin http://www.mhhe.com/biosci/cellmicro/kalthoff/sample_ch04.pdf

8. 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

9. 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

10. 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

11. 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

13. 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.

14. Models Analyzed Using the standard bands the following models were analyzed: –linear/linear (Using Log MW) ….y=mx+b –quadratic……………….....y=a+bx+cx^2 –cubic……………………...y=a+bx+cx^2+dx^3 –special cubic……………...y=a+bx+cx^3 –special log………………. y=a+bx+clnx

15. Model/Analysis For each of the five different models, predicted values, standard deviations, and the R-squared was calculated.

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

17. 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

18. Why did the Cubic Model best predict the data set for Gel #1 and Gel #2? GEL #1 GEL #2

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

20. 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.

21. 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.

22. Molecular Weight (Band) Comparison

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

24. 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.

25. 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.

26. 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.