Sperm Enzyme Preparation (Group 6)

Comparing all Gradient Gel Standards

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

Gathering Data:

Gels 1 and 2

Hypothesis (Part One)

Standards will be reproducible.

Hypothesis (Part Two)

Has more proteins than

Standards

Measured three timesCalculated relative mobilitiesCalculated mean measurementsBest fit mathematical model

Mathematical Models

Table of Molecular Weights and the Log

of the Molecular Weights:

Protein

Molecular Weight

sLog Molecular

Weights

Myosin 200000.00 5.30

beta-Galactosida

se 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

Best Model Determination

Rank Models tried StdError t-inv Error

1 y=a+bX+cX^2+dX^3 0.0729 2.5706 0.1873

2 y=a+bX+cX^3 0.0935 2.4469 0.2287

3 y=a+bX 0.0968 2.3646 0.2288

4 y=a+bX+cX^2+(lnX)^2 0.089 2.5706 0.2289

5 y=a+bX+cX^2 0.0974 2.4469 0.2383

6 y=a+bX+c(lnX)+d(lnX)^2 0.0957 2.5706 0.2461

Model with its CI

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

4.0

4.5

5.0

5.5

RmMean

logM

wlogMw = 5.96754 - 5.56606 RmMean

+ 9.56238 RmMean**2 - 6.92265 RmMean**3

S = 0.0729144 R-Sq = 98.5 % R-Sq(adj) = 97.6 %

Regression

95% CI

Regression Plot

Comparison Chart

3.5000

3.7000

3.9000

4.1000

4.3000

4.5000

4.7000

4.9000

5.1000

5.3000

5.5000

0 0.2 0.4 0.6 0.8 1

Rm

Lo

gM

w PreVs.RmMean

StdLogMwVs.RmMean

StdRmGel1

StdRmGel2

The Reaction

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

Comparing Unknowns to Standards

Vitelline Envelope control Sperm enzyme control Vitelline Envelope + sperm enzyme (t=0 min) Vitelline Envelope + enzyme (t=5 min) Vitelline Envelope + enzyme (t=10 min) Vitelline Envelope + enzyme (t=15 min) Vitelline Envelope + enzyme (t=30 min)

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

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

Results: In gel 1, the vitelline envelope remains the same

throughout the experiment.

VE control t=0 t=5min t=10min t=15min t=30min

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

Results: Vitelline envelope breaks up as the bands

become greater.VE control t=0 t=5min t=10min t=15min t=30min 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

Results: StandardsLogMw Vs. Rm

3.5

3.7

3.9

4.1

4.3

4.5

4.7

4.9

5.1

5.3

5.5

0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95

Rm

Lo

gM

w

Rm1

Rm2

Rm3

Rm4

Rm5

Rm6

Rm7

Rm8

Rm9

Rm10

Rm11

Rm12

Rm13

Rm14

Rm15

Rm16

Rm17

Rm18

Rm19

Rm20

Rm21

Rm22

mean

Conclusion:

Standards are reproducible.Sperm enzyme breaks up vitelline envelope.

– Number of bands increase.– Small proteins are being detected.