Tritium calculation

[RoC]

Hi there,

I would like to carry out an enzyme activity assay for the galactosyltransferase enzyme in the Golgi of CHO cells. I found a paper with a protocol using tritium labelled UDP-Glucose as donor substrate. I've tried to contact the authors for more details but have gotten no reply. As its my first time working with radioactivity I would like to know if my dilution calculations of radiolabeled glucose are ok or are totally incorrect? Your advice is greatly appreciated.

 

 

The protocol suggests using 0.5 mM Glucose at 50,000 dpm / nm in a 25 ul assay. I take it the dilution must first by made up for the radiolabeled glucose with non-labeled glucose before addition to the final assay volume. I was thinking of making up a 1ml volume stock at 5 mm UDP-Glucose at 50,000 dpm /nm.

 

 

Calculations for UDP-[3H]Glucose in GalTI assay:

 

Radioactive Concentration (RAC): 1000 µCi/ml

Specific Activity (SA): 15 Ci/mmol

Volume of final Assay mix: 25 µl

Desired concentration of final diluted mix: 0.5 mM

 

Specific Activity:

Equation to convert Ci/mmol to DPM/fmol: DPM/fmol = [specific activity (Ci/mmol) x [2.22 x 1012 DPM/Ci] x [mmol/1012 fmol] = SA x 2.22

SA = 15 Ci/mmol DPM/fmol = SA x 2.22 = 15 x 2.22 = 33.3 DPM/fmol

= 33.3 x 106 = DPM/nmol

= 33,300,000 DPM/nmol

 

Protocol suggested to use UDP-[3H]Glucose at 50,000 DPM/nmol

 

Nominal Stock Concentration

[Radioligand] = RAC/SA = (1000 µCi/ml / 15 Ci/mmol) 1 Ci/106 µCi = 66.6 x 10-6 mmol/ml

= 66.6 µmol

= 66.6 µM

66.6 µM = 66600 nmol / L therefore

 

1ml of stock = 66.6 nmol

1ul = 0.666 nmol

And if 33.3 x 106 dpm/nmol (from specific activity)

then there is 2.2 x 107 dpm / ul of hot stock

 

 

Dilution

 

What volume (ul’s) of radioactive UDP-Glucose is required to be added a 1 ml solution of 5 mM UDP-Glucose to get a concentration of 5 mM UDP-Glucose at 50,000 dpm / nm?

 

5mM stock concentration in 1 mL volume for addition to assay

5mM / 1ml = 5 x 103 nmol / ml

5,000 nmol / ml @ 50,000 dpm / nm = 2.5 x 108 dpm

 

2.5 x 108 ÷ 2.2 x 107 = 11.3 ul of hot stock to be added to 1 ml of 5 mm cold UDP-GlcNAc to get a concentration of 50,000 dpm / nm.

[cypress]

Hi there,

I would like to carry out an enzyme activity assay for the galactosyltransferase enzyme in the Golgi of CHO cells. I found a paper with a protocol using tritium labelled UDP-Glucose as donor substrate. I've tried to contact the authors for more details but have gotten no reply. As its my first time working with radioactivity I would like to know if my dilution calculations of radiolabeled glucose are ok or are totally incorrect? Your advice is greatly appreciated.

 

 

The protocol suggests using 0.5 mM Glucose at 50,000 dpm / nm in a 25 ul assay. I take it the dilution must first by made up for the radiolabeled glucose with non-labeled glucose before addition to the final assay volume. I was thinking of making up a 1ml volume stock at 5 mm UDP-Glucose at 50,000 dpm /nm.

 

 

Calculations for UDP-[3H]Glucose in GalTI assay:

 

Radioactive Concentration (RAC): 1000 µCi/ml

Specific Activity (SA): 15 Ci/mmol

Volume of final Assay mix: 25 µl

Desired concentration of final diluted mix: 0.5 mM

 

Specific Activity:

Equation to convert Ci/mmol to DPM/fmol: DPM/fmol = [specific activity (Ci/mmol) x [2.22 x 1012 DPM/Ci] x [mmol/1012 fmol] = SA x 2.22

SA = 15 Ci/mmol DPM/fmol = SA x 2.22 = 15 x 2.22 = 33.3 DPM/fmol

= 33.3 x 106 = DPM/nmol

= 33,300,000 DPM/nmol

 

Protocol suggested to use UDP-[3H]Glucose at 50,000 DPM/nmol

 

Nominal Stock Concentration

[Radioligand] = RAC/SA = (1000 µCi/ml / 15 Ci/mmol) 1 Ci/106 µCi = 66.6 x 10-6 mmol/ml

= 66.6 µmol

= 66.6 µM

66.6 µM = 66600 nmol / L therefore

 

1ml of stock = 66.6 nmol

1ul = 0.666 nmol

And if 33.3 x 106 dpm/nmol (from specific activity)

then there is 2.2 x 107 dpm / ul of hot stock

 

I see one error here: I get 2.2x10^6 dpm/ul it looks like you did not shift the decimal point far enough since 1ul = 0.067 nmol.

 

Dilution

 

What volume (ul’s) of radioactive UDP-Glucose is required to be added a 1 ml solution of 5 mM UDP-Glucose to get a concentration of 5 mM UDP-Glucose at 50,000 dpm / nm?

 

5mM stock concentration in 1 mL volume for addition to assay

5mM / 1ml = 5 x 103 nmol / ml

 

5,000 nmol / ml @ 50,000 dpm / nm = 2.5 x 108 dpm

 

2.5 x 108 ÷ 2.2 x 107 = 11.3 ul of hot stock to be added to 1 ml of 5 mm cold UDP-GlcNAc to get a concentration of 50,000 dpm / nm.

 

So I get 110 ul. I don't see any other issue in your calculations except the minor error of adding 110 ul to a 1000 ul solution. I would think that you would want to add the cold solution to 110 ul of hot stock to make 1 ml total.

[RoC]

I see one error here: I get 2.2x10^6 dpm/ul it looks like you did not shift the decimal point far enough since 1ul = 0.067 nmol.

 

 

 

So I get 110 ul. I don't see any other issue in your calculations except the minor error of adding 110 ul to a 1000 ul solution. I would think that you would want to add the cold solution to 110 ul of hot stock to make 1 ml total.

 

 

That's great. Thanks for that. Much appreciated.

RoC