Metabolic Labeling of Nascent Proteins
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Corresponding Organization : Memorial Sloan Kettering Cancer Center
Other organizations : Hong Kong University of Science and Technology, University of Hong Kong, The University of Texas MD Anderson Cancer Center, Cancer Genetics (United States), Columbia University Irving Medical Center
Variable analysis
- Addition of the methionine analog L-azidohomoalanine (AHA; Invitrogen) to the culture medium
- Addition of MG-132 (Sigma, M7449; 5 μM) to the culture medium
- Incorporation of AHA into nascent proteins
- Biotin cross-linking of AHA-labeled nascent proteins
- Capture of biotin-cross-linked nascent proteins using streptavidin-coated Dynabeads
- Separation of AHA-labeled, biotin-cross-linked, streptavidin-precipitated proteins by SDS–PAGE
- Isogenic Nalm-6 cells
- Culture of cells in fresh medium for 24 hrs
- Washing of cells twice with PBS
- Resuspension of cells in methionine-free RPMI 1640 medium (Gibco) supplemented with 10% FCS for 30 min
- No positive or negative controls were explicitly mentioned in the protocol.
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