Puromycin Labeling of Nascent Peptides

The assay was performed as described (Ogami et al. 2017 (link)). Plasmid transfected HEK293T cells were treated with 1 µg/mL puromycin for 30 min to label nascent peptides. Control cells were incubated with 10 µg/mL cycloheximide for 10 min before puromycin treatment. The puromycin-labeled proteins were then resolved by SDS-PAGE and detected by Western blotting using antipuromycin antibody (Kerafast EQ 0001).

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Tsai Y.L., Coady T.H., Lu L., Zheng D., Alland I., Tian B., Shneider N.A, & Manley J.L. (2020). ALS/FTD-associated protein FUS induces mitochondrial dysfunction by preferentially sequestering respiratory chain complex mRNAs. Genes & Development, 34(11-12), 785-805.

Publication 2020

EQ 0001

Antibody Assay Cells Cycloheximide Peptides Plasmid Proteins Puromycin Sds page

Other organizations : Columbia University, Rutgers, The State University of New Jersey

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Variable analysis

independent variables

Puromycin treatment (1 µg/mL for 30 min)
Cycloheximide treatment (10 µg/mL for 10 min before puromycin)

dependent variables

Puromycin-labeled proteins detected by Western blotting

control variables

Plasmid transfected HEK293T cells

controls

Positive control: Puromycin treatment to label nascent peptides
Negative control: Cycloheximide treatment to inhibit protein synthesis before puromycin labeling

Annotations

Based on most similar protocols

Optimize puromycin concentration and incubation time to achieve robust labeling of nascent peptides (Protocol 1: 25 μM O‐propargyl‐puromycin for indicated time).

Validate the method using positive controls, such as incubating cells with cycloheximide to inhibit protein synthesis and prevent puromycin incorporation (Input Protocol: 10 μg/mL cycloheximide for 10 min).

Ensure complete permeabilization of cells to allow efficient detection of puromycin-labeled proteins (Protocol 1: Permeabilized with TBST (TBS with 0.2% Triton X‐100)).

Perform CuAAC (copper(I)-catalyzed azide-alkyne cycloaddition) detection of the incorporated O‐propargyl‐puromycin as described in Liu et al. (2012) to enhance signal detection (Protocol 1).

Record the incubation temperature, as it can affect the efficiency of puromycin incorporation (Protocol 1: 43°C).

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