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

• Pig heart NADP+-IDH in 50% glycerol (Sigma # I-2002)
• Dilute 60 ml in 1 ml of Assay Buffer (above)
• Test for appropriate levels of activity and adjust as needed
• Keep on ice at all times

Isocitrate Stock Solution (4.6 mM)

• 11.9 mg of DL-isocitrate (Sigma # I-1252 ) in 10 mL of Assay Buffer (above)

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)

• 11 mg of NADP+ (Sigma # N-0505) in 5 mL of Assay Buffer

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)

• 38 mg in 1 mL of water
• Final concentration when 10 ml is added to 200 ml of assay solution: 5 mM.

NaCl Solution (5 M)

• Dissolve 0.293 g in 1 mL of assay buffer

Survey of IDH in Different Organisms

• Obtain fresh materials
• Homogenize 3-5 g in equal volume of Assay Buffer
• Freeze (preferably at -70° C)
• Thaw before use
• Centrifuge to remove cellular debris
• Use supernatant as IDH source

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