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| This protocol can be used to measure the amount of incorporation of 35S-Methionine or other radiolabeled amino acids into cellular protein following a metabolic labeling procedure. A protein extract needs to be made before commencing this protocol. |
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1. Spot 1 to 2 μl of radiolabeled protein extract (CAUTION! see Hint #2) onto a small pieces of Whatman glass microfiber GF/C or hardened ashless 541 filter paper (see Hint #1).
2. Allow filters to dry at room temperature or under a heat lamp.
3. Add filter samples to about 60 ml of boiling 10% TCA (CAUTION! see Hint #2).
4. Boil for 5 min.
5. Wash the filters in ice-cold 5% TCA for 3 min.
6. Repeat the wash in ice-cold 5% TCA.
7. Wash the filters in ice-cold 95% Ethanol for 1 min.
8. Repeat the wash in ice-cold 95% Ethanol.
9. Allow the filter samples to dry.
10. Add the filters to the scintillation vial along with a toluene-based scintillation fluid.
11. Count the samples in a scintillation counter.
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| 95% (v/v) Ethanol |
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| 5% (v/v) TCA |
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| 10% (v/v) TCA |
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| 100% TCA Stock |
| Dissolve in 227 ml ddH2O
500 g Trichloroacetic Acid (TCA)
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Scintillation Fluid
Trichloroacetic Acid
Ethanol
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1. For larger sample volumes, increase the size of the filter paper.
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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