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Protocol

Test Plate Experiment

Protocol

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TEST PLATE

1

2

3

4

5

6

7

8

9

10

11

12

A

Batch Ref (VP)

141171

141201

613

151280

141361

1231

1095

141374

Batch QC (GW)

141345

1091

B

Batch QC (GW)

1207

141232

587

141335

1116

Batch Ref (VP)

1030

1197

151068

1014

141112

C

687

141318

Batch QC (GW)

987

141229

1205

151009

141352

141364

629

Batch Ref (VP)

481

D

1211

151217

131276

141202

151178

Batch QC (GW)

592

Batch Ref (VP)

141346

1226

151019

301

E

1175

1111

1236

Batch Ref (VP)

534

141130

Batch QC (GW)

141091

141348

141337

141362

141372

F

141365

Batch Ref (VP)

151187

1262

141440

141236

1189

1034

Batch QC (GW)

638

141088

1132

G

532

1210

141387

141078

1171

Batch Ref (VP)

141217

Batch QC (GW)

1018

1163

1237

141370

H

1133

151034

141099

151190

Batch QC (GW)

1167

141353

1113

1078

141070

1229

Batch Ref (VP)

 

 

n

 

Batch Ref

8

Ventricular Pool

Healthy Control

51

Group 1

AD/MCI

14

Group 2

PD/PDI-MCI/LBD

15

Group 3

Batch QC

8

Golden West

 

 

Row 1

 

Row 2

 

Row 3

 

Row 4

 

Row 5

 

Row 6

 

Row 7

 

Row 8

 

Batch Ref (VP)

B1VP-01

Batch QC (GW)

B1QC-02

687

ADC1-21

1211

ADC1-31

1175

ADC1-41

141365

ADC1-51

532

ADC1-61

1133

ADC1-71

141171

ADC1-01

1207

ADC1-11

141318

ADC1-22

151217

ADC1-32

1111

ADC1-42

Batch Ref (VP)

B1VP-06

1210

ADC1-62

151034

ADC1-72

141201

ADC1-02

141232

ADC1-12

Batch QC (GW)

B1QC-03

131276

ADC1-33

1236

ADC1-43

151187

ADC1-52

141387

ADC1-63

141099

ADC1-73

613

ADC1-03

587

ADC1-13

987

ADC1-23

141202

ADC1-34

Batch Ref (VP)

B1VP-05

1262

ADC1-53

141078

ADC1-64

151190

ADC1-74

151280

ADC1-04

141335

ADC1-14

141229

ADC1-24

151178

ADC1-35

534

ADC1-44

141440

ADC1-54

1171

ADC1-65

Batch QC (GW)

B1QC-08

141361

ADC1-05

1116

ADC1-15

1205

ADC1-25

Batch QC (GW)

B1QC-04

141130

ADC1-45

141236

ADC1-55

Batch Ref (VP)

B1VP-07

1167

ADC1-75

1231

ADC1-06

Batch Ref (VP)

B1VP-02

151009

ADC1-26

592

ADC1-36

Batch QC (GW)

B1QC-05

1189

ADC1-56

141217

ADC1-66

141353

ADC1-76

1095

ADC1-07

1030

ADC1-16

141352

ADC1-27

Batch Ref (VP)

B1VP-04

141091

ADC1-46

1034

ADC1-57

Batch QC (GW)

B1QC-07

1113

ADC1-77

141374

ADC1-08

1197

ADC1-17

141364

ADC1-28

141346

ADC1-37

141348

ADC1-47

Batch QC (GW)

B1QC-06

1018

ADC1-67

1078

ADC1-78

Batch QC (GW)

B1QC-01

151068

ADC1-18

629

ADC1-29

1226

ADC1-38

141337

ADC1-48

638

ADC1-58

1163

ADC1-68

141070

ADC1-79

141345

ADC1-09

1014

ADC1-19

Batch Ref (VP)

B1VP-03

151019

ADC1-39

141362

ADC1-49

141088

ADC1-59

1237

ADC1-69

1229

ADC1-80

1091

ADC1-10

141112

ADC1-20

481

ADC1-30

301

ADC1-40

141372

ADC1-50

1132

ADC1-60

141370

ADC1-70

Batch Ref (VP)

B1VP-08

 

B1VP-01

ADC1-01

ADC1-02

ADC1-03

ADC1-04

ADC1-05

ADC1-06

ADC1-07

ADC1-08

B1QC-01

ADC1-09

ADC1-10

B1QC-02

ADC1-11

ADC1-12

ADC1-13

ADC1-14

ADC1-15

B1VP-02

ADC1-16

ADC1-17

ADC1-18

ADC1-19

ADC1-20

ADC1-21

ADC1-22

B1QC-03

ADC1-23

ADC1-24

ADC1-25

ADC1-26

ADC1-27

ADC1-28

ADC1-29

B1VP-03

ADC1-30

ADC1-31

ADC1-32

ADC1-33

ADC1-34

ADC1-35

B1QC-04

ADC1-36

B1VP-04

ADC1-37

ADC1-38

ADC1-39

ADC1-40

ADC1-41

ADC1-42

ADC1-43

B1VP-05

ADC1-44

ADC1-45

B1QC-05

ADC1-46

ADC1-47

ADC1-48

ADC1-49

ADC1-50

ADC1-51

B1VP-06

ADC1-52

ADC1-53

ADC1-54

ADC1-55

ADC1-56

ADC1-57

B1QC-06

ADC1-58

ADC1-59

ADC1-60

ADC1-61

ADC1-62

ADC1-63

ADC1-64

ADC1-65

B1VP-07

ADC1-66

B1QC-07

ADC1-67

ADC1-68

ADC1-69

ADC1-70

ADC1-71

ADC1-72

ADC1-73

ADC1-74

B1QC-08

ADC1-75

ADC1-76

ADC1-77

ADC1-78

ADC1-79

ADC1-80

B1VP-08

 

 

 

MAKE SURE HEATING PLATES/BLOCKS ARE AT CORRECT TEMP

 

 

 

MATERIALS:

  • Pooled ventricular CSF; 8 60 ul aliquots
  • GW CSF; 3 250 ul aliquots
  • BCA reagent; 22,000 ul total
    • 21,560 ul of reagent A
    • 440 ul of reagent B
  • 1M iodoacetamide (IAA) (1 plate)
  • 1M DL-dithiothreitol (DTT) (1 plate)
  • 50 mM Tris, pH 8.5
    • 30 mL
      • 1.5 mL 50 1M Tris + 28.5 mL water; pH to 8.5
  • 2% SDS
    • Dilute 20% SDS with Tris pH 8.5
    • 2,000 ul 20% SDS in 18,000 ul Tris
  • Resyn Biosciences MagResyn Hydroxyl particles
  • 100% Acetonitrile (ACN)
  • 70% Ethanol
    • 200 mL
  • 95% Acetonitrile
    • 300 mL
  • 100 ng/ul enolase
    • 1,750 ul (0.2 ug/ul) aliquot in freezer 3
    • Thaw, add 1,600 ul of it to reservoir with 1,600 ul of lysis buffer 
  • 50 mM ammonium bicarbonate (ABC)
  • Pierce Porcine trypsin; 3 100-ug vials
    • Add 2,000 ul of 50 mM ABC to each vial to get working concentration of 0.05 ug/ul
  • HPLC grade 100% formic acid
  • 500 fmol Pierce Retention Time Calibrant
    • Dilute 5 pmol PRTC (stock) to 500 fmol

 

Materials cont'd

  • Labeled binding, wash, and elution plates
  • Labeled reservoirs

 

 

 

METHODS:

 

Protein Assay

Thaw everything, and put on ice in order in 4 buckets

Perform BCA assay on the to determine protein concentration

200 ul of BCA reagent into each well of the assay plate

*3 rows per plate, then bring it to the 37 degree room, so 4 total plates

14 of 2% SDS into the 4 sample wells

Add 2 ul of sample to the wells

Add 16 ul of each standard to the wells

Pipette up and down to mix and heat for 30 min at 37 C

Use Varioskan flash to obtain photometric reading of the plates

 

Calculations

Whichever sample has the lowest concentration will be the baseline, so all other samples will be brought up to this volume with DPBS

So the amount of 2% SDS added to every sample will be the same

 

sample

conc

amt need for 25 ug

amt DPBS needed to bring to lowest conc vol

ul enolase

vol 2% SDS to add

1M IAA

1M DTT

vol for 70% final conc of ACN

Batch Ref (VP)

.896

27.9

6.39

8

34.29

3.06

1.6

 

141171

.941

26.56

7.73

8

34.29

3.06

1.6

 

141201

1.204

20.77

13.52

8

34.29

3.06

1.6

 

613

.769

32.5

1.79

8

34.29

3.06

1.6

 

151280

.769

32.5

1.79

8

34.29

3.06

1.6

 

141361

.914

27.35

6.94

8

34.29

3.06

1.6

 

1231

.91

27.49

6.8

8

34.29

3.06

1.6

 

1095

.846

29.55

4.74

8

34.29

3.06

1.6

 

141374

.819

30.52

3.77

8

34.29

3.06

1.6

 

Batch QC (GW)

.95

26.31

7.98

8

34.29

3.06

1.6

 

141345

.973

25.7

8.59

8

34.29

3.06

1.6

 

1091

.878

28.48

5.81

8

34.29

3.06

1.6

 

Batch QC (GW)

.86

29.08

5.21

8

34.29

3.06

1.6

 

1207

.882

28.33

5.96

8

34.29

3.06

1.6

 

141232

.887

28.19

6.1

8

34.29

3.06

1.6

 

587

.869

28.78

5.51

8

34.29

3.06

1.6

 

141335

.742

33.69

.6

8

34.29

3.06

1.6

 

1116

1.122

22.28

12.01

8

34.29

3.06

1.6

 

Batch Ref (VP)

.946

26.44

7.85

8

34.29

3.06

1.6

 

1030

.882

28.33

5.96

8

34.29

3.06

1.6

 

1197

1.

25.

9.29

8

34.29

3.06

1.6

 

151068

.937

26.69

7.6

8

34.29

3.06

1.6

 

1014

.914

27.35

6.94

8

34.29

3.06

1.6

 

141112

.869

28.78

5.51

8

34.29

3.06

1.6

 

687

8.416

2.97

31.32

8

34.29

3.06

1.6

 

141318

.869

28.78

5.51

8

34.29

3.06

1.6

 

Batch QC (GW)

1.054

23.71

10.58

8

34.29

3.06

1.6

 

987

.991

25.23

9.06

8

34.29

3.06

1.6

 

141229

.882

28.33

5.96

8

34.29

3.06

1.6

 

1205

.864

28.93

5.36

8

34.29

3.06

1.6

 

151009

.878

28.48

5.81

8

34.29

3.06

1.6

 

141352

.742

33.69

.6

8

34.29

3.06

1.6

 

141364

.824

30.36

3.93

8

34.29

3.06

1.6

 

629

.91

27.49

6.8

8

34.29

3.06

1.6

 

Batch Ref (VP)

.919

27.22

7.07

8

34.29

3.06

1.6

 

481

.792

31.57

2.72

8

34.29

3.06

1.6

 

1211

.946

26.42

7.87

8

34.29

3.06

1.6

 

151217

.907

27.57

6.72

8

34.29

3.06

1.6

 

131276

1.132

22.08

12.21

8

34.29

3.06

1.6

 

141202

.828

30.18

4.11

8

34.29

3.06

1.6

 

151178

.824

30.36

3.93

8

34.29

3.06

1.6

 

Batch QC (GW)

.892

28.02

6.27

8

34.29

3.06

1.6

 

592

.926

26.98

7.31

8

34.29

3.06

1.6

 

Batch Ref (VP)

.941

26.56

7.73

8

34.29

3.06

1.6

 

141346

.961

26.02

8.27

8

34.29

3.06

1.6

 

1226

.804

31.1

3.19

8

34.29

3.06

1.6

 

151019

.892

28.02

6.27

8

34.29

3.06

1.6

 

301

1.015

24.64

9.65

8

34.29

3.06

1.6

 

1175

.877

28.49

5.8

8

34.29

3.06

1.6

 

1111

.819

30.54

3.75

8

34.29

3.06

1.6

 

1236

.838

29.82

4.47

8

34.29

3.06

1.6

 

Batch Ref (VP)

.985

25.37

8.92

8

34.29

3.06

1.6

 

534

.755

33.12

1.17

8

34.29

3.06

1.6

 

141130

1.01

24.76

9.53

8

34.29

3.06

1.6

 

Batch QC (GW)

.877

28.49

5.8

8

34.29

3.06

1.6

 

141091

.819

30.54

3.75

8

34.29

3.06

1.6

 

141348

.838

29.82

4.47

8

34.29

3.06

1.6

 

141337

.863

28.98

5.31

8

34.29

3.06

1.6

 

141362

1.064

23.5

10.79

8

34.29

3.06

1.6

 

141372

.961

26.02

8.27

8

34.29

3.06

1.6

 

141365

.941

26.56

7.73

8

34.29

3.06

1.6

 

Batch Ref (VP)

1.157

21.61

12.68

8

34.29

3.06

1.6

 

151187

.907

27.57

6.72

8

34.29

3.06

1.6

 

1262

.843

29.65

4.64

8

34.29

3.06

1.6

 

141440

.99

25.25

9.04

8

34.29

3.06

1.6

 

141236

.917

27.27

7.02

8

34.29

3.06

1.6

 

1189

.799

31.29

3.

8

34.29

3.06

1.6

 

1034

.833

30.

4.29

8

34.29

3.06

1.6

 

Batch QC (GW)

.912

27.42

6.87

8

34.29

3.06

1.6

 

638

.892

28.02

6.27

8

34.29

3.06

1.6

 

141088

.912

27.42

6.87

8

34.29

3.06

1.6

 

1132

.961

26.02

8.27

8

34.29

3.06

1.6

 

532

.802

31.17

3.12

8

34.29

3.06

1.6

 

1210

.729

34.29

.

8

34.29

3.06

1.6

 

141387

.844

29.63

4.66

8

34.29

3.06

1.6

 

141078

.875

28.57

5.72

8

34.29

3.06

1.6

 

1171

.823

30.38

3.91

8

34.29

3.06

1.6

 

Batch Ref (VP)

.875

28.57

5.72

8

34.29

3.06

1.6

 

141217

.943

26.52

7.77

8

34.29

3.06

1.6

 

Batch QC (GW)

.88

28.4

5.89

8

34.29

3.06

1.6

 

1018

.786

31.79

2.5

8

34.29

3.06

1.6

 

1163

.828

30.19

4.1

8

34.29

3.06

1.6

 

1237

.974

25.67

8.62

8

34.29

3.06

1.6

 

141370

.891

28.07

6.22

8

34.29

3.06

1.6

 

1133

.833

30.

4.29

8

34.29

3.06

1.6

 

151034

.932

26.82

7.47

8

34.29

3.06

1.6

 

141099

1.

25.

9.29

8

34.29

3.06

1.6

 

151190

1.208

20.69

13.6

8

34.29

3.06

1.6

 

Batch QC (GW)

1.

25.

9.29

8

34.29

3.06

1.6

 

1167

1.208

20.69

13.6

8

34.29

3.06

1.6

 

141353

.953

26.23

8.06

8

34.29

3.06

1.6

 

1113

.969

25.81

8.48

8

34.29

3.06

1.6

 

1078

.901

27.75

6.54

8

34.29

3.06

1.6

 

141070

.938

26.67

7.62

8

34.29

3.06

1.6

 

1229

1.083

23.08

11.21

8

34.29

3.06

1.6

 

Batch Ref (VP)

1.

25.

9.29

8

34.29

3.06

1.6

 

 

 

 

Protein denaturation

To each well add a volume of 2% SDS equivalent to the volume of CSF added

Pipette up and down gently

Add DPBS to bring up samples to the volume equivalent to the most dilute sample

 

Sample preparation

Based on the concentrations, pull 25 ug and add to plate

Add 8 ul of 100 ng/ul enolase

 

 

Reduction and alkylation

Volumes of reducing and alkylating agents will be calculated after the sample and buffer volumes are determined (above)

Add ____ ul of 1M DTT to each well, pipette up and down gently

Heat the plate at 95C for 10 min

Allow to cool

Add ____ ul of 1M IAA to each well

Pipette up and down gently

Incubate at room temperature in the dark for 30 minutes

 

Protein precipitation

Add 12.5 ul of the Hydroxyl beads (at their stock concentration of 20 ug/ul) to each well

Add _____ ul of 100% ACN to each well (70% final concentration) to precipitate proteins

Allow to sit for 10 minutes at room temperature

 

NOTE: You cannot freeze the samples at this point--the hydroxyl beads will rupture when frozen.

 

Wash and digestion plate preparation

Wash plates (deep well):

  • Washes 1-3; 1 mL 95% ACN
  • Washes 4-5; 1 mL EtOH

 

Digestion/elution plate (deep well):

  • 100 ul 50 mM ABC + 50 ul of 0.05 ug/ul Pierce Porcine trypsin (1:10 enzyme:protein ratio)

 

Kingfisher automated clean-up and digestion

Turn on Kingfisher and bring up adapted Resyn protocol (see attached)

Make sure binding plate volume has been adjusted

Add plates to instrument as indicated by the program

Hit "run"

 

The automated clean-up and digestion will take about 90 minutes.

 

Quenching the digestion

Add 5.4 ul of 100% formic acid to quench

Freeze (-80C) or dry down in speed vacuum

In my case, I transferred the samples from the 96 well plate and into 8 individual 1.5 mL tubes (NOT lo-bind)

 

  Attached Files  
   
 test-plate-protocol-experimental-notes.pdf

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