Plasmids were linearized with BamHI and then purified using QiaQuick Purification columns (Qiagen). The Sf-21 TnT Coupled Transcription/Translation System was utilized for all translation assays (Promega). In this system, linearized plasmid is added to the extracts and RNA is synthesized by T7 polymerase and subsequently translated. Total reaction volume was 10 μl containing: 6.7 μl of Sf-21 cell extract (Promega), 0.3 μl of l -[35S]- methionine/cysteine (PerkinElmer, >1000 Ci/mmol) and 1 μg linearized plasmid DNA. For edeine experiments, in vitro transcribed RNAs were incubated in Sf-21 cell extracts. Each reaction mixture was incubated at 30°C for 2 h and then resolved on a 15% SDS-PAGE. Gels were dried and radioactive bands were monitored via phosphoimager analysis. For quantitation of bands, the number of methionines and cysteines were accounted for and normalized for each protein. For T2A containing constructs (37 (link)), luciferase activity was monitored using a Dual-Luciferase reporter assay system (Promega) and an Infinite M1000 PRO microplate reader (Tecan).
L 35s methionine cysteine
Manufactured by PerkinElmer
The L-35S-methionine/cysteine is a radioactive amino acid used for labeling proteins. It contains the radioactive isotope sulfur-35, which can be incorporated into methionine and cysteine residues during protein synthesis. This product is commonly used in various research applications that involve the study of protein expression, translation, and trafficking.
Automatically generated - may contain errors
Lab products found in correlation
Sf 21 cell extract, by Promega (1 mentions)
Qiaquick purification columns, by Qiagen (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Cyclone plus storage phosphor system, by PerkinElmer (1 mentions)
Criterion xt bis tris gel, by Bio-Rad (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
3 protocols using l 35s methionine cysteine
1
In vitro Protein Translation Assay
2
Metabolic Labeling of HEK-MOR Cells
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At various time points after morphine treatment, HEK-MOR cells with the density of 5 × 105 cells/cm2 were washed with PBS and incubated in methionine-free DMEM (Invitrogen), but containing 30 μCi/ml L-35S-methionine/cysteine (Perkin Elmer) for 1 h.
3
Pulse Labeling of CADA Substrates in CHO Cells
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For the pulse labeling of the CADA substrates, CHO-K1 cells were seeded at a cell density of 2 × 105 cells/ml in Ham's F-12 medium (Gibco) supplemented with 10% fetal bovine serum and allowed to adhere overnight. The next day, cells were transfected with plasmid DNA using Lipofectamine LTX (Invitrogen) according to the manufacturer’s protocol and incubated overnight at 37 °C. Cells were pretreated with 10 μM CADA and/or 200 nM MG132 (Sigma) for 1 h prior to a 45-min starvation for methionine and cysteine. Next, cells were pulsed for 30 min with L-35S-methionine/cysteine (PerkinElmer) and chased for 45 min at 37 °C. All steps were performed under constant CADA (10 μM) and MG132 (200 nM) pressure. Chased cells were then washed with ice-cold medium and lysed with NP-40 buffer as described previously. Radiolabeled proteins were pulled down using V5-trap agarose beads (Chromotek) as described in the manufacturer's protocol. The pulled proteins were next separated with SDS-PAGE on 4 to 12% Criterion XT Bis–Tris gel (Bio-Rad) using 2-(N-morpholino)ethanesulfonic acid buffer, detected by phosphor imaging and quantified (Cyclone Plus storage phosphor system; PerkinElmer).
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