All the experiments were performed following the protocol described in ref. 45 (link) with some modifications. MEFs were seeded in six-well plates with 106 cells per well in 1 ml complete DEMEM, and treated with 1 μg ml−1 puromycin solution (Sigma) for 10 min at 37 °C and 5% CO2 (=pulse). The cells were washed twice with pre-warmed complete medium, resuspended in 1 ml of the same medium and incubated for 50 min at 37 °C and 5% CO2 (=chase). The cells were washed twice with cold PBS, and harvested. They were then washed once more by centrifugation at 4 °C in cold PBS/0.1% BSA, and pellets were incubated in 30 μl of staining mix (mouse IgG2 anti-puromycin antibody (12D10) diluted 1/50 in PBS/BSA) for 30 min. The cells were washed twice with PBS/BSA and incubated in 30 μl of PE-labelled anti-mouse IgG (Invitrogen) diluted 1/100 in PBS/BSA. The cells were washed and resuspended in 200 μl PBS/BSA and analysed by FACS (fluorescence-activated cell sorting).
Puromycin solution
Manufactured by Merck Group
Puromycin solution is a cell culture reagent commonly used as a selection marker in genetic engineering applications. It functions as an antibiotic, inhibiting protein synthesis in eukaryotic cells. This solution can be used to select for cells that have successfully incorporated a puromycin resistance gene, allowing for the isolation and propagation of genetically modified cell lines.
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Lab products found in correlation
3 protocols using puromycin solution
1
Puromycin Labeling of Nascent Proteins
2
Reagent Acquisition for IGF-IR and InsR Signaling
3
Reagent Acquisition for IGF-IR and InsR Signaling
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IGF-I, IGF-II and insulin were purchased from Gemini and Eli Lily, respectively. Puromycin solution was purchased from Sigma-Aldrich. Geneticin (G418 sulfate) solution was purchased from Life Technologies. Humanized anti-IGF1R monoclonal antibody HuEM164 was generously provided by Immunogen Inc. Anti-InsR monoclonal antibody, alpha subunit clone 83-7 was purchased from EMD Millipore. S961 peptide was generously provided by Novo Nordisk, Denmark (27 (link)).
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