Click chemistry labeling protocol was performed as previously described [56] (link) and adopted to Sf9 cells as follows. Sf9 cells expressing 6×His-tagged gephyrin were labeled with 100 µM 17-ODYA (Cayman Chemicals) for 6 h. Cells were washed with PBS and resuspended into 400 µl of cold lysis buffer (100 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1% Tritox X-100) containing protease inhibitor complete cocktail (Roche). Cells were briefly sonicated and incubated on ice for 30 min under frequent vortexing. After centrifugation of unbroken cells, the supernatant was subjected to chloroform/methanol precipitation and resuspended in 2% SDS, 50 mM Tris/Cl, 5 mM EDTA, pH 7.4. Click chemistry reaction began with 23 µl of cell lysates containing ∼2 mg/ml protein, 0.5 µl 5 mM biotin-PEG-N3 prepared in DMSO, 0.5 µl of 50 mM Tris(2-carboxyethyl)phosphine hydrochloride prepared freshly, and 0.5 µl 10 mM Tris[(1-benzyl-1H-1,2,3-triazol-4y1)methyl-1]amine prepared in DMSO/t-butanol 1∶4 v/v; was vortexed for 5 s, followed by adding 0.5 µl 50 mM CuSO4/5H20 (freshly prepared); was vortexed again; and was incubated for 1 h at room temperature in the dark. All chemicals were from Sigma. Affinity purification of biotinoylated proteins with Neutravidin beads was performed as described for the ABE assay.
17 odya
Manufactured by Cayman Chemical
Sourced in United States
17-ODYA is a laboratory reagent used in biochemical research. It is a carboxylic acid derivative that can be utilized in various analytical procedures. The core function of 17-ODYA is to serve as a research tool for investigating cellular processes and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Cuso4, by Merck Group (2 mentions)
Biotin azide, by Cayman Chemical (2 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
2 bromopalmitate 2 bp, by Merck Group (1 mentions)
17 octadecynoic acid, by Cayman Chemical (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
2 bromopalmitate, by Merck Group (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Wwl70, by Cayman Chemical (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
X tremegene 9, by Roche (1 mentions)
14 15 eet, by Cayman Chemical (1 mentions)
11 12 eet, by Cayman Chemical (1 mentions)
Transwell plate, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
9 protocols using 17 odya
1
Click Chemistry Labeling of Gephyrin in Sf9 Cells
2
Probing Spike Protein Lipidation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expressions of Spike and its various mutants were carried out by transfecting HEK293T cells with 3 μg DNA and PEI. After 36 h the media was replaced with 1 ml of DMEM conditioned with 1%, fatty acid free, Bovine Serum Albumin (BSA; Sigma). The same media contained 50 μM 2-bromopalmitate (2BP; Sigma) solubilized in DMSO for 2BP experiments. Post 2 h, cells were fed ∼0.1 mM saponified (41 (link)) fatty acid alkynes 17-Octadecynoic Acid (17-ODYA; Cayman Chemical) or 15-hexadecynoic acid (15-yne; Avanti Polar) dissolved in DMEM condition with 20%, fatty acid free, BSA. After 6 hours cells were harvested into microfuge tubes with two washes of 1× PBS.
3
Lipid Modification Proteomics Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipofectamine 2000, Lipofectamine RNAiMax, sodium dedocyl sulfate (SDS) solution, L-azidohomoalanine (L-AHA), Alexa Fluor 488-azide (AF488-az), Alexa Fluor 647-alkyne (AF647-alk), TRIzol reagent, and Prolong Gold Antifade Mountant with DAPI were purchased from Life Technologies (Burlington, ON). X-tremeGENE 9 was purchased from Roche (Indianapolis, IN). Palmostatin B was purchased from Merck Scientific (Billerica, MA). Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), Triton-X 100 (TX-100), sodium deoxycholate, CuSO4, palmitic acid, and 2-bromopalmitate were obtained from Sigma-Aldrich (St. Louis, MO). 17-ODYA, C75, WWL70, and RHC-80267 were purchased from Cayman Chemical (Ann Arbor, MI). HDFP, C83, and C115 were gifts from Dr. Brent Martin (University of Michigan), and FP-rhodamine was generously provided by Dr. Benjamin Cravatt (Scripps Institute).
4
Cytotoxicity and Migration Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture medium (RPMI 1640 medium) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). The ELISA kits for 11,12-EET and 14,15-EET were obtained from Detroit R&D (Detroit, MI, USA). 11,12-EET, 14,15-EET, and 17-ODYA were purchased from Cayman Chemical (Ann Arbor, MI, USA). 3-(4, 5-dimethyl-2-thiazy1)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and DMSO were obtained from Sigma Chemical Co. (St. Louis, MO, USA). PI and Annexin V/FITC detection kits were purchased from Bender Medsystems Inc. (Burlingame, CA, USA). Antibodies against CyclinD1, Bax, Bcl-2, MMP-2, and MMP-9 were purchased from Epitomics Inc. (Burlingame, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-rabbit IgG) were purchased from KPL (Gaithersburg, MA, USA). Enhanced chemiluminescence reagents were purchased from Pierce, Inc. (Rockford, IL, USA). The Transwell plates were obtained from Corning Costar (Cambridge, MA, USA), and the Matrigel was purchased from BD Biosciences (Bedford, MA, USA).
5
Shedding and Labeling of TNF-R1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The labeling protocol was adapted from [25 ]. In brief, to shed TNF-R1 from the cell surface and to trigger transport from the PM cells were washed in PBS and incubated in the presence of 150 μM histamine for 3 h in FCS free medium at cell culture conditions as adapted from Wang et al [26 (link)]. Histamine treated and untreated cells were then incubated for 16 h in the presence of 100 μM 17-ODYA (#90270, Cayman), followed by membrane sedimentation as described for acylRAC. The resulting pellet was resuspended in 150 μl 25 mM HEPES [pH 7.4], 25 mM NaCl, 0.5% Triton X-100, PIC. The click reaction was made up fresh with the final concentrations: 500 μM biotin-azide (#13040, Cayman)), 2 mM CuCO4, 0.2 mM TBTA (#678937, Sigma Aldrich) and 4 mM ascorbic acid (fresh) in a total volume of 200 μl. After 2 h incubation at RT, proteins were acetone precipitated and then resuspended in 500 μl 50 mM TRIS-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1% Triton X-100, 1 mM EDTA, 0.25% Na-deoxycholate. 20 μl Streptavidin-microbeads (#130–048-102, Miltenyi) were added and incubated overnight at 4 °C. After purification via μColums (Miltenyi) and elution using SDS-Sample buffer containing β-mercaptoethanol, 15 μl were used for SDS-PAGE/WB.
6
Biochemical Pulldown Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DiIC12 was purchased from Life Technology. NBD-PE was purchased from Avanti. anti-myc AlexaFluora-647, anti-biotin, and anti-myc were purchased from Cell Signaling Technologies. anti-myc AlexaFluora-647 labeled antigen binding fragment (Fab) was purchased from Promega. DTT, Tris(2-carboxyethyl)phosphine (TCEP), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) were purchased from Research Products International; DTT was prepared fresh for every use. Paraformaldehyde (PFA) was purchased as a 16% stock solution from Electron Microscopy Sciences. CaCl2, NaCl, CuCl2, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), anti-myc magnetic beads, and propidium iodide were purchased from Fisher Scientific. 17-ODYA and biotin-azide were purchased from Cayman Chemicals. HeLa cell lines were acquired from American Type Culture Collection. Primary RSCs were a generous gift from the laboratory of Bruce Carter at Vanderbilt University (Nashville, TN).
7
Metabolic Labeling and Inhibition Assay
8
Palmitoylation Profiling Using Click Chemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SCs, HEK293T and other cells transfected with the indicated plasmids or siRNAs were incubated in medium supplemented with 100 μM 17-octadecynoic acid (17-ODYA), an alkyne-containing palmitate analog (Cayman Chemical, 90,270) for 6–8 h. Cells were then washed with phosphate-buffered saline (PBS) and briefly sonicated in detergent-free lysis buffer. Membrane fractions were made by ultracentrifugation and then pellets were lysed in lysis buffer with 1% (v:v) Triton X-100. An aliquot (94 μl) of each cleared lysate was reacted with 6 μl of freshly premixed click chemistry reagent (final 100 μM biotin-azide, 1 mM Tris [2-carboxyethyl] phosphine, 100 μM Tris-[benzyltriazolylmethyl] amine, and 1 mM CuSO4) [34 (link)]. Reaction mixtures were incubated for 1 h at room temperature (RT), and then 2 μl of 0.5 M EDTA was added to terminate the reactions. Unreacted biotin was quenched by the addition of an excess of 1 M Tris, pH 8.0. Biotinylated (palmitoylated) proteins were then affinity isolated using streptavidin beads by incubation at 4 °C for 2 h. The beads were washed 3 times with PBS containing 0.5% Triton X-100 and treated with sample buffer for western blot.
9
Palmitic Acid Analogue Labeling and Enrichment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transiently transfected HEK293E cells were treated with 25 µm of the palmitic acid analogue, 17‐ODYA (Cayman Chemical) or mock treated with an equal volume of DMSO, 24 h after transfection. The 17‐ODYA labelling was allowed to continue for 6 h at 37°C under standard culture conditions. Cell pellets were collected and lysed in 1% Triton X‐100/50 mm Tris‐Cl pH 7.4/150 mm NaCl/protease inhibitor cocktail (Roche) for 30 min at 37°C with shaking. The click chemistry reaction was then performed as previously described (Martin and Cravatt, 2009 ). Briefly, 2 mg of each protein sample was incubated with the following chemicals in PBS: 100 µm biotin‐azide (Life Technologies), 1 mm Tris(2‐carboxyethyl)phosphine (TCEP; Sigma‐Aldrich), 100 µm Tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA, Sigma‐Aldrich) dissolved in DMSO/tert‐butanol (20%/80%), and 1 mm CuSO4 (Sigma‐Aldrich). The reaction was allowed to continue for 1.5 h at room temperature with shaking. Biotinylated proteins were affinity purified by incubating with a 50 µl bed volume of streptavidin‐agarose (Thermo Scientific) for 2 h at room temperature with rotation. Proteins were eluted by incubating with 100 µl of 2% SDS/50 mm Tris‐Cl pH 7.4/5 mm EDTA for 5 min at 95°C with shaking.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!