3. Which of the following molecules is useful in protein phosphorylation studies?
a. α-32P ATP
b. β-32P ATP
c. γ-32P ATP
d. ATP-γ-S
γ-32P ATP is the correct answer for protein phosphorylation studies. Protein kinases transfer the γ-phosphate from ATP to substrate proteins, so radiolabeling at this position enables detection of phosphorylation via autoradiography or scintillation counting.
Option Analysis
Protein kinases catalyze the transfer of the terminal γ-phosphate group from ATP to serine, threonine, or tyrosine residues on target proteins, making the γ-position critical for labeling studies.
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a. α-32P ATP: The α-phosphate forms part of ATP’s backbone and remains in ADP after γ-transfer, so it labels nucleic acids (e.g., PCR, transcription) but not protein phosphorylation.
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b. β-32P ATP: The β-phosphate is released as pyrophosphate (PPi) with the γ-phosphate during kinase reactions, preventing its incorporation into proteins.
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c. γ-32P ATP: The γ-phosphate is directly transferred to proteins, allowing precise tracking of kinase activity and phosphorylated sites in assays.
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d. ATP-γ-S: This thiophosphate analog supports thiophosphorylation (resistant to phosphatases) but is not standard for routine radiolabeling studies due to slower transfer and specialized detection needs.
Protein phosphorylation studies rely on γ-32P ATP as the gold standard for tracking kinase activity, where the radiolabeled γ-phosphate transfers directly to protein substrates. This technique, vital for CSIR NET Life Sciences exams, quantifies phosphorylation by measuring incorporated 32P via gel electrophoresis and autoradiography.
Radiolabeled ATP in Kinase Assays
Kinase reactions use [γ-32P] ATP because only the terminal phosphate group attaches to proteins, enabling sensitive detection down to nanomolar levels. Assays incubate kinase, substrate, and labeled ATP, then separate phosphorylated products to count radioactivity, confirming site-specific modifications.
Why Not Other Options?
α-32P ATP suits DNA/RNA labeling since its phosphate stays in the chain, while β-32P ATP’s label exits as PPi. ATP-γ-S creates stable thiophosphates for phosphatase-resistant studies but requires non-radioactive detection like Phos-tag gels.
| Molecule | Phosphate Transferred | Use Case | Limitation in Phosphorylation Studies |
|---|---|---|---|
| α-32P ATP | No (stays in ADP) | Nucleic acid labeling | Does not label proteins |
| β-32P ATP | No (released as PPi) | Transcription initiation studies | Label not incorporated |
| γ-32P ATP | Yes (to protein) | Standard kinase assays | None for this purpose |
| ATP-γ-S | Yes (thiophosphate) | Irreversible phosphorylation | Slower kinetics, non-radioactive |
