Equipment and Materials for IDH Enzyme Lab

Making Solutions for IDH Enzyme Lab

Microplate Readers (1 or 2) - BioRad Model 3550UV

Printers (2)

Microcentrifuge

Computers

Graphing program

Water bath and thermometer

P-20, P-200, P-1000 micropipets

8-Channel Multi-Pipets

Yellow tips

Blue tips

96-well plates

Buffer troughs

1.5 mL microfuge tubes, racks

Sharpies

Assay Buffer

Pig heart IDH

NADP+ solutions

Isocitrate solutions

EDTA solution

Metal ion solutions

Trash receptacles


Instructors Notes
Making Solutions

An excellent assignment is to have students verify molarities of these solutions.


Assay Buffer, pH 8.5

 Tris-HCl  7.32 g
 Tris Base  18.6 g
 MgCl2  203 mg
 Distilled water  to 1 liter


Enzyme Solution


Isocitrate Stock Solution (4.6 mM)


Concentrations of Isocitrate for Experiment 2-3 (1 mL each; about 100 assays)

 Tube

 mM*

µl of Stock
Isocitrate Sol

 µl of Assay Buffer

 1  0.23  1000  0
 2  0.058  252  742
 3  0.029  125  875
 4  0.019  83  917
 5  0.01  43  957

*Final concentration when 10 ml is added to 200 ml of assay solution.


NADP+ Stock solution (2.88 mM)


Dilute Concentrations of NADP+ (1 mL each; about 100 assays)

 Tube

 mM*

µl of Stock
NADP+ Stock Sol

 µl of Assay Buffer

 1 0.144  1000  0
 2 0.072  500  500
 3 0.036  250  750
 4 0.0216  150  850
 5 0.0144  100  900

* Final concentration when 10 ml is added to 200 ml of assay solution.


Tris-HCl Buffers for pH Experiment

Stock Solutions A, B, and C
 A  0.2 M Tris Base  24.2 g in one liter
 B  0.2 M HCl  16.7 mL of 12 N HCl in one liter
 C  1.0 mM MgCl2  2.03 g in 10 mL

Mix 50 mL of A + X mL of B + distilled water to 200 mL final volume

Add 20 ml of C to each 200 mL solution

 X

 Approximate pH  Actual pH **
 5.0 9.0  
8.1 8.8  
12.2 8.6  
16.5 8.4  
21.9 8.2  
26.8 8.0  
32.5 7.8  
38.4 7.6  
41.4 7.4  
44.2 7.2  

**Determine.
Phosphate buffers may be used for a wider range of pH values.


Divalent Metal Ion Stock Solutions (100 mM)

 Metal*

 Sigma Catalog #

 mg/mL

 MgCl2 M-9272 20.3
MnCl2 M-3634  19.8
CuCl2 C-6641 13.5
ZnCl2 Z-3500 13.6

* Final concentration when 10 ml is added to 200 ml of assay solution: 5 mM.


EDTA Stock Solution (100 mM)


NaCl Solution (5 M)


Survey of IDH in Different Organisms


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© Copyright 2000 Department of Biology, Davidson College, Davidson, NC 28036
Send comments, questions, and suggestions to: macampbell@davidson.edu