Anti-FLAG M2 (#F1804) and Anti-FLAG M2-HRP (#A8592) were purchased from Sigma-Aldrich and anti-Src antibody (#MA5-15120) was from Thermo Scientific. p-Src (Y416) (#2113), p-Abl (Y412) (#2865), p-Tyr-100 (#9411), p-Tyr-1000 (#8954), anti-GAPDH (#14C10), Myc Tag (mouse #2276 and rabbit #71D10), p-AKT (T308) (#13038), p-AKT (S473) (#4060), p70 S6 kinase (#9202), and phospho-p70 S6K (T389) (#9234) primary antibodies were purchased from Cell Signaling Technologies (CST). Secondary antibodies Goat anti-Mouse-HRP (#31430) and Goat anti-Rabbit-HRP (#31460) were from Thermo Scientific. Alexa Fluor 488 anti-mouse (#A11029) and Alexa Fluor 647 anti-rabbit (#A21245) were from Life Technologies. All primary antibodies were used at a 1:1000 dilution and secondaries at 1:5000. Protease inhibitor cocktail (#P8340), Phosphatase inhibitor cocktail 2 (#P0044) and 3 (#P5726) were purchased from Sigma-Aldrich. NeuCode labeling reagents L-Lysine:2HCl (3,3,4,4,5,5,6,6-D8, 98%) (#DLM-2641-0) and L-Lysine:2HCl (13C6, 99%, 15N2, 99%) (#CNLM-291-H-0.25) were from Cambridge Isotopes. Rapamycin was from Cayman Chemical and M-PER extraction reagent (#78501) was from Thermo Scientific. All restriction enzymes and DNA polymerases were purchased from NEB (Ipswich, MA). Oligonucleotides and gBlocks Gene Fragments were purchased from IDT and all constructs were verified by DNA sequencing (Quintara Biosciences).
L lysine 2hcl 13c6
Manufactured by Cambridge Isotopes
L-Lysine:2HCl (13C6) is a stable isotope labelled amino acid product. It is used as a research tool in applications such as metabolic studies and tracer experiments.
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Lab products found in correlation
Anti rabbit alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse hrp, by Thermo Fisher Scientific (1 mentions)
Phospho p70s6k t389, by Cell Signaling Technology (1 mentions)
M per extraction reagent, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
P src y416, by Cell Signaling Technology (1 mentions)
Pakt t308, by Cell Signaling Technology (1 mentions)
P tyr 100, by Cell Signaling Technology (1 mentions)
P tyr 1000, by Cell Signaling Technology (1 mentions)
P70 s6 kinase, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti flag m2 hrp, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
Pakt s473, by Cell Signaling Technology (1 mentions)
Dialyzed fbs, by Thermo Fisher Scientific (1 mentions)
L arginine hcl, by Cambridge Isotopes (1 mentions)
2 protocols using l lysine 2hcl 13c6
1
Anti-FLAG and Phospho-Specific Antibody Protocol
2
Stable Isotopic Labeling in Cell Culture
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Before isotopic labeling, cultures were grown to confluency. Once cells ceased cell division and were contact inhibited, they were maintained in a quiescent state for 4 days. Subsequently, cells were acclimated to the labeling media (EMEM) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin for 4 days. After 4 additional days in the adaptation media, cultures were introduced to the minimum essential medium for stable isotopic labeling in cell culture (SILAC) (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.13 g/l and 0.0904 g/l, respectively, 15% dialyzed FBS (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. After 0, 2, 4, and 6 days of labeling, cells were harvested and washed with PBS, and cell pellets were frozen before further analysis.
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