Sperm Enzyme Preparation (Group 6)

1
Sperm Enzyme Preparation (Group 6)

• Comparing all Gradient Gel Standards

2
Experiment Objective

• Compare all given gradient gel standards and use
  their relative mobilities to create the best fit
  mathematical model.
• Use best model to compare two gel samples

3
Gathering Data
Gels 1 and 2
4
Hypothesis (Part One)

• Standards will be reproducible.

5
Hypothesis (Part Two)
Has more proteins than
6
Standards

• Measured three times
• Calculated relative mobilities
• Calculated mean measurements
• Best fit mathematical model

7
Mathematical Models

• Table of Molecular Weights and the Log of the
  Molecular Weights

Protein Molecular Weights Log Molecular Weights
Myosin 200000.00 5.30
beta-Galactosidase 116250.00 5.07
Phosphorlyase B 97400.00 4.99
Serum albumin 66200.00 4.82
Ovalbumin 45000.00 4.65
Carbonic anhydrase 31000.00 4.49
Trypsin inhibitor 21500.00 4.33
Lysozyme 14400.00 4.15
Aprotinin 6500.00 3.81
8
Best Model Determination
Rank Models tried StdError t-inv Error
1 yabXcX2dX3 0.0729 2.5706 0.1873
2 yabXcX3 0.0935 2.4469 0.2287
3 yabX 0.0968 2.3646 0.2288
4 yabXcX2(lnX)2 0.089 2.5706 0.2289
5 yabXcX2 0.0974 2.4469 0.2383
6 yabXc(lnX)d(lnX)2 0.0957 2.5706 0.2461
9
Model with its CI
10
Comparison Chart
11
The Reaction

• The expansion of the Vitelline Envelope due to
  contact with sperm over a 30 minute time period.

12
Comparing Unknowns to Standards

• Vitelline Envelope control
• Sperm enzyme control
• Vitelline Envelope sperm enzyme (t0 min)
• Vitelline Envelope enzyme (t5 min)
• Vitelline Envelope enzyme (t10 min)
• Vitelline Envelope enzyme (t15 min)
• Vitelline Envelope enzyme (t30 min)

13
Results

• Sperm enzyme controls between gels 1 and 2 were
  similar.

SpermEz SpermEz
5.324899 5.232962
5.226104 4.893098
4.938703 4.844403
4.880003 4.805563
4.805201 4.746631
4.74529 4.660919
4.641404 4.593366
4.556522 4.544095
4.322521 4.396276
  4.194351
  3.937239
14
Results

• Vitelline envelope controls are similar between
  gels one and two.

VE-control VE-control
5.025182 4.97541
4.836222 4.858922
4.603148 4.675516
4.556547 4.595335
4.507308 4.541261
4.369169 4.474135
  4.373088
15
Results

• In gel 1, the vitelline envelope remains the same
  throughout the experiment.

VE control t0 t5min t10min t15min t30min
5.04546 5.06347 5.04636 5.04628 5.00209 5.04128
4.85299 4.86845 4.86472 4.85728 4.80612 4.84932
4.63242 4.70620 4.70417 4.69804 4.62657 4.70021
4.59116 4.64479 4.63953 4.63564 4.57749 4.64055
4.54808 4.60985 4.59991 4.59237 4.52437 4.56039
4.42873 4.53289 4.53481 4.52491 4.40474 4.34733
16
Results

• Vitelline envelope breaks up as the bands become
  greater.

VE control t0 t5min t10min t15min t30min Enz Control
4.99457 5.03009 5.02913 5.02049 5.30636 5.01015 5.25677
4.87586 4.91907 4.91500 4.92597 5.00280 4.92357 4.91052
4.69793 4.84398 4.84474 4.85187 4.90688 4.84508 4.86121
4.62546 4.70464 4.76903 4.78914 4.84393 4.78317 4.82238
4.57774 4.64180 4.70679 4.70738 4.78363 4.70430 4.76481
4.51925 4.56680 4.61328 4.65542 4.70127 4.63763 4.68454
4.43210   4.53940 4.62324 4.64289 4.56924 4.62371
      4.55644 4.60213 4.45604 4.58022
      4.48425 4.54159 3.96395 4.45204
      3.96678 4.46121   4.27900
        3.96145   4.05928
17
Results Standards
18
Conclusion

• Standards are reproducible.
• Sperm enzyme breaks up vitelline envelope.
• Number of bands increase.
• Small proteins are being detected.