Protein gel lanes were excised and cut into five pieces. Gel pieces were washed in pure H2O followed by washes in NH4HCO3 (25 mM, Sigma-Aldrich) and CH3CN (50%, Thermo Fisher Scientific). Further, gel pieces were dehydrated in CH3CN (100%) before incubation (56°C, 45 min) in NH4HCO3 (25 mM) and DTT (10 mM, Sigma-Aldrich). Subsequently, samples were incubated (RT, 45 min) in NH4HCO3 (25 mM) containing iodoacetamide (55 mM, Sigma-Aldrich), followed by washing (RT, 5 min) in NH4HCO3 (25 mM) and CH3CN (50%) and dehydration in CH3CN (100%). Then, each piece was incubated (ice, 30 min) in 15 μl of trypsin (12.5 ng/ml, Thermo Fisher Scientific) and NH4HCO3 (50 mM). The excess of trypsin was removed and gel pieces were covered with NH4HCO3 (50 mM) and incubated over night at 37°C. The tryptic peptides were extracted the following day by incubating twice in HCOOH (5%, Thermo Fisher Scientific) and CH3CN (50%) followed by incubation in CH3CN (100%). Peptides from gel pieces that originated from the same sample were merged and completely dried in a speed vac. The dried peptides were resuspended in 40 μl of HCOOH (0.1%) prior to MS analysis and equal volumes (10 μl) were injected in the mass spectrometer for shotgun and targeted MS analysis.
Nh4hco3
Manufactured by Thermo Fisher Scientific
Sourced in China, United States, Germany, Belgium
NH4HCO3 is an inorganic compound with the chemical formula (NH4)HCO3. It is a white, crystalline solid that is soluble in water. NH4HCO3 is commonly used as a pH regulator, buffer, and in various chemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (3 mentions)
Acetonitrile, by Thermo Fisher Scientific (3 mentions)
Urea, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Nh4hco3, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Ch3cn, by Thermo Fisher Scientific (1 mentions)
Ch3cn, by Merck Group (1 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (1 mentions)
Rna extraction kit, by Solarbio (1 mentions)
Protein extraction kit, by Solarbio (1 mentions)
Pbs buffer, by Thermo Fisher Scientific (1 mentions)
Protein loading buffer, by Beyotime (1 mentions)
Coomassie brilliant blue r 250, by Solarbio (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Hplc grade water, by Thermo Fisher Scientific (1 mentions)
7 protocols using nh4hco3
1
Tryptic Peptide Extraction for Mass Spectrometry
2
Extraction and Characterization of Cannabis Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh seeds of C. sativa var. Longma 5, which was a commercial variety from Heilongjiang Province, China (one of the major provinces in hemp cultivation), were obtained from Ms. Mu (General manager of Heilongjiang Heike Technology Co. Ltd). The chemicals including NaOH and NaCl were purchased from Hongyan Co. (Tianjin, China); NH4HCO3, acetonitrile, dithiothreitol, acetonitrile, formic acid, RNase-free DNase I and PBS buffer were purchased from Thermo Fisher Scientific (San Jose, CA, USA); n-hexane was purchased from Aladdin Co. (Shanghai, China); HCl was purchased from Kermel Co.(Tianjin, China); KOD enzyme was purchased from Toyobo Co. (Osaka, Japan); pGEM-T plasmid was purchased from Takara Co. (Kyoto, Japan); 5X protein loading buffer was purchased from Beyotime Co. (Shanghai, China); Coomassie brilliant blue R-250 was purchased from Solarbio Co. (Beijing, China). The kits including protein extraction kit and RNA extraction kit were purchased from Solarbio Co. (Beijing, China) and Tiangen Co. (Beijing, China), respectively. All reagents used in the study were of analytical grade or higher purity.
3
Gel-based Protein Digestion and Peptide Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The excised protein gel band was cut to small particles with a spot picker and washed twice with HPLC-grade water (Fisher) and incubated with 1:1 v/v of 0.1 NH4HCO3 (Sigma) for 15 min. Wash solution was removed and HPLC-grade acetonitrile (Fisher) was then added to cover the gel particles for 1 hr, acetonitrile removed and gel particles were rehydrated in 0.1 M NH4HCO3 for 10 min. An equal volume of ACN was then added for a 1:1 v/v of 0.1 NH4HCO3/ACN. After 10 min all liquid was removed and the gel particles dried under vacuum then reduced with 10 mM dithiothreitol (DTT, Thermo Scientific) and alkylated with 55 mM iodoacetamide (GE Healthcare) in 0.1 M NH4HCO3. After reduction and alkylation, gel particles were washed and trypsin digestion for 24 h at 37 °C. Resulting peptides were extracted by addition of a 25 mM NH4HCO3, 5% formic acid and acetonitrile (2:1:1). The tryptic digest mixture was desalted with a C18 ZipTip (Millipore) and eluted into 60% methanol containing 0.1% formic acid. Pooled digested peptides were dried and re-suspended in formic acid: water: acetonitrile (0.1:95:5) for liquid chromatographic separation and electrospray ionization mass spectrometry (LC-ESI MS) analysis.
4
Tryptic In-Gel Protein Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gels were cut into 1 cubic millimeters blocks and washed with ddH2O in centrifuge tubes. 100 μl pH 8.0 50% (v:v) ACN (Fisher, USA) plus 25 mM NH4HCO3 (Fisher, USA) were used to destain the gels for 15 minutes, repeat 3 times until all the color faded. Immerse the gel blocks into 30 μl 100% ACN for dehydration until they turn white, drain the water out at room temperature. Tryptic in-gel digestion was conducted by the addition of 8 μl trypsin (0.005 mg ml−1) (Promega, USA) at 37 °C for 16 h.
5
Peptide Synthesis Protocol Methodology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All Fmoc amino acids, reagents, and solvents were used without purification. Fmoc amino acids and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) were purchase from AAPPTec, Bachem, Chem-Impex, ChemPep, and PurePep. The Rink amide ChemMatrix resin (catalog number: 7-600-1310) was purchased from Biotage. The 2-chlorotrityl chloride (2-CTC) resin (catalog number: 150301) were purchased from ChemPep. N,N-dimethylformamide (DMF), CH2Cl2, MeCN, MeOH, Et2O, AcOH, Ac2O, N,N-diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), NH4HCl, NH4HCO3, urea, NaOH, 80% hydrazine hydrate, 2-mercaptoethanol were purchased from Fisher Scientific. Piperidine, triisopropylsilane (TIPS), hydroxybenzotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC), 2,2′-dithiodipyridine (DTDP), 2,2′-dithiobis (5-nitropyridine) (DTNP), I2, Pd(PPh3)4, 1,3-dimethylbarbituric acid (DMBA), glucose, sodium ascorbate were purchased from Sigma Aldrich. 4-carboxy-3-fluorophenylboronic acid was purchased from Alfa Aesar. Agilent 6120 Quadrupole LC‒MS system was used to acquire the LC chromatograms and mass spectra of samples with Luna® 5 μm C18 100 Å (50 × 2 mm) column (Phenomenex, CA, United States) at 0.4 ml/min with 5% of a H2O/MeCN + 0.1% TFA solution for 1 min followed by a linear gradient from 5% to 95% of a H2O/MeCN + 0.1% TFA solution over 5 min.
6
Proteomic Sample Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dithiothreitol (DTT), iodoacetamide
(IAA), and HEPES were purchased from Sigma-Aldrich Chemie, Steinheim,
Germany, and urea and NH4HCO3 from Acros Organics,
Geel, Belgium. Trypsin, protease inhibitor, dimethyl sulfoxide, Dulbecco’s
Modified Eagle Medium, fetal bovine serum albumin, and the Coomassie
Plus (Bradford) Assay Kit were from Thermo Scientific, Waltham, MA.
Tris(hydroxymethyl)aminomethane hydrochloride (Tris), LC–MS
grade water, acetonitrile, and formic acid were obtained from Fisher
Scientific, Geel, Belgium. RapiGest SF Surfactant was purchased from
Waters Corporation, Milford, MA.
(IAA), and HEPES were purchased from Sigma-Aldrich Chemie, Steinheim,
Germany, and urea and NH4HCO3 from Acros Organics,
Geel, Belgium. Trypsin, protease inhibitor, dimethyl sulfoxide, Dulbecco’s
Modified Eagle Medium, fetal bovine serum albumin, and the Coomassie
Plus (Bradford) Assay Kit were from Thermo Scientific, Waltham, MA.
Tris(hydroxymethyl)aminomethane hydrochloride (Tris), LC–MS
grade water, acetonitrile, and formic acid were obtained from Fisher
Scientific, Geel, Belgium. RapiGest SF Surfactant was purchased from
Waters Corporation, Milford, MA.
7
Mass Spectrometry-based Proteomic Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gibco Dulbecco’s Modified
Eagle Medium, high glucose (DMEM), PierceTM Trypsin protease MS grade,
protease inhibitor, DMSO, Corning Premium Fetal Bovine Serum (FBS),
Invitrogen accutase, phosphate buffered saline (PBS), doxycycline,
isobutyl-1-methylxanthine (IBMX), adrenocorticotropin hormone (ACTH),
α-melanocyte stimulating hormone (α-MSH), γ-melanocyte
stimulating hormone (γ-MSH), UPLC–MS grade water, acetonitrile,
and formic acid were purchased from Thermo Fisher Scientific (Waltham,
MA). Dithiothreitol (DTT), iodoacetamide (IAA), HeLa digest, 4-(3-butoxy-4-methodybenzyl)
imidazolidone (Ro 20-1724), and HEPES were obtained from Sigma-Aldrich
Chemie (Steinheim, Germany). Urea and NH4HCO3 were obtained from Acros Organics (Geel, Belgium). Tris(hydroxymethyl)aminomethane
hydrochloride (Tris), LC–MS grade water, acetonitrile, and
formic acid were obtained from Fisher Scientific (Geel, Belgium).
RapiGest SF Surfactant was purchased from Waters Corporation (Milford,
MA). The cAMP-GloTM Assay including cAMP-GloTM Lysis Buffer, cAMP-GloTM
reaction buffer, Protein Kinase A, Kinase-Glo substrate, and Kinase-Glo
Buffer were from Promega Corporation (Madison, WI). Stock solutions
of all ligands were prepared in 0.1 mM DMSO.
Eagle Medium, high glucose (DMEM), PierceTM Trypsin protease MS grade,
protease inhibitor, DMSO, Corning Premium Fetal Bovine Serum (FBS),
Invitrogen accutase, phosphate buffered saline (PBS), doxycycline,
isobutyl-1-methylxanthine (IBMX), adrenocorticotropin hormone (ACTH),
α-melanocyte stimulating hormone (α-MSH), γ-melanocyte
stimulating hormone (γ-MSH), UPLC–MS grade water, acetonitrile,
and formic acid were purchased from Thermo Fisher Scientific (Waltham,
MA). Dithiothreitol (DTT), iodoacetamide (IAA), HeLa digest, 4-(3-butoxy-4-methodybenzyl)
imidazolidone (Ro 20-1724), and HEPES were obtained from Sigma-Aldrich
Chemie (Steinheim, Germany). Urea and NH4HCO3 were obtained from Acros Organics (Geel, Belgium). Tris(hydroxymethyl)aminomethane
hydrochloride (Tris), LC–MS grade water, acetonitrile, and
formic acid were obtained from Fisher Scientific (Geel, Belgium).
RapiGest SF Surfactant was purchased from Waters Corporation (Milford,
MA). The cAMP-GloTM Assay including cAMP-GloTM Lysis Buffer, cAMP-GloTM
reaction buffer, Protein Kinase A, Kinase-Glo substrate, and Kinase-Glo
Buffer were from Promega Corporation (Madison, WI). Stock solutions
of all ligands were prepared in 0.1 mM DMSO.
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!