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alpha-Ketoglutarate (alpha-KG)

-Ketoglutarate (a-KG) is used in the determination of transaminases, malate dehydrogenase, glutamate dehydrogenase, urea and ammonia.



1.Source: Disodium salt
   Form: Crystalline powder 
   Molecular Weight: 190.1 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20 C (-4 F) 
   Protein: 70% 
   Catalog No.: 215J0000 
$5.00/g


1. 0.1M Triethanolamine buffer/substrate, pH 7.6: 1.86 g TEA• HCl with 80 ml distilled water. Adjust pH to 7.6 with 1M NaOH-Na2. Adjust volume to 100 ml with distilled water.
2. 14 mM NADH: 10 mg NADH-Na2 with 1 ml distilled water.
3. 3 M Ammonium chloride: 16.05 g NH4Cl with 100 ml distilled water.
4. Glutamate dehydrogenase, from beef liver: 20 mg protein/ml (120 U/mg).



1. Dissolve 25 mg Ketoglutarate in 25 ml distilled water in a volumetric flask.
2. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25° C.
3. Into a cuvette, pipette the following:
BLANK SAMPLE
Buffer (1) 2.80 ml 2.80 ml
NADH (2) 0.05 ml 0.05 ml
NH4Cl (3) 0.15 ml 0.15 ml
distilled H2O 0.05 ml - - -
sample - - - 0.05 ml
Mix and read absorbance A1.
4. Start the reaction by adding 0.01 ml of GLDH to both the blank and sample. Mix and read absorbance A2.



A = (A1 - A2)sample - (A1 - A2)blank
Concentration of -KG = X A (mg/ml sample solution)
Concentration of -KG-Na2 H2• 3H2 O = X A (mg/ml sample solution)
% -KG = X 100
Total Vol. = 3.06 ml
190.1 = MW of -KG
Sample Vol. = 0.05ml

 

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