Protein bands were excised from gels, finely chopped, and washed with 200 µl of 50 mM ammonium bicarbonate (Bio‐Rad Laboratories). Gel pieces were destained using solution of 25 mM NH4HCO3/ACN, 1/1, v/v (Sigma‐Aldrich) following by acetonitrile (ACN). Proteins were reduced by 10 mM DTT at 56°C for 1 hr and then alkylated by 55 mM iodoacetamide at room temperature for 45 min in the dark. In‐gel digestion was performed by overnight‐incubation at 37°C with 1 µg of sequencing‐grade trypsin (Roche) in 50 mM ammonium bicarbonate. Then, the supernatants were collected and acidified with formic acid up to a final concentration of 1%. And the peptides were extracted following the method of previous published paper (Colzani et al., 2016 ).
Ammonium bicarbonate
Manufactured by Roche
Sourced in United States, Germany
Ammonium bicarbonate is a laboratory chemical compound with the chemical formula (NH4)HCO3. It is a colorless, crystalline solid that is commonly used as a pH buffer, leavening agent, and in various analytical and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Sequencing grade trypsin, by Roche (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ammonium bicarbonate, by Bio-Rad (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Urea, by Roche (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
L dopa, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Sequencing grade chymotrypsin, by Roche (1 mentions)
Sequencing grade modified trypsin, by Roche (1 mentions)
Modified porcine trypsin, by Promega (1 mentions)
Sonopuls hd3200, by Bandelin (1 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (1 mentions)
Cp02 automated dry pulverizer, by Covaris (1 mentions)
5 protocols using ammonium bicarbonate
1
In-Gel Tryptic Protein Digestion
2
In-Gel Digestion of AGE-BSA and ALE-HSA
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Polypeptide bands corresponding to AGE-BSA, obtained by incubation with ribose or glucose, and ALE-HSA incubated with MDA and retained by VC1, were excised from gels. After a brief wash with 100 μl of 50 mM ammonium bicarbonate (Sigma Aldrich), gel pieces were incubated with 10 mM dithiothreitol at 56 °C for 30 min and then with 55 mM iodoacetamide (Sigma Aldrich) at room temperature for 45 min in the dark. In-gel digestion of AGE-BSA and ALE-HSA was performed by overnight-incubation at 37 °C with 1 µg of sequencing-grade trypsin (Roche) dissolved in 50 mM ammonium bicarbonate. ALE-HSA was also subjected to a second digestion by a sequencing-grade chymotrypsin (1 µg) for 7 h at 25 °C in the presence of calcium chloride (10 mM). Peptide mixtures were extracted by a 10 min-incubation with extraction solution (acetonitrile/trifluoroacetic acid/water; 30/3/67; v/v/v) and by an additional 10 min-incubation with 100% acetonitrile. The two extracts were combined and dried in a vacuum concentrator (Martin Christ.). Digested peptide mixtures were then dissolved in an appropriate volume of 0.1% formic acid for mass spectrometry (MS) analysis.
3
Melanogenesis Inhibition Assay with L-DOPA
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The following reagents were sourced from Sigma-Aldrich (Saint Louis, MI, USA): L-DOPA (Cat #D9628); urea (Cat #U5378); thiourea (Cat #T7875); C7BzO (Cat #C0856); Dulbecco’s Modified Eagle Medium (DMEM; Cat#D6429) (New York, NY, USA); cOmplete™ Protease Inhibitor Cocktail (Cat #11697498001) (Roche, USA); tris(2-carboxyethyl)phosphine (TCEP; Cat #C4706); ammonium bicarbonate (Cat #A6141); Empore™ Extraction Disk Cartridge C18 (Cat #66873-U) (Supelco, Bellefonte, PA, USA); acetonitrile hypergrade for LC–MS LiChrosolv® (Cat #1.00029) (Truganina, Australia); triflouroacetic acid (Cat #T6508); tyrosinase (Cat #T3824); boric acid (Cat #1.00165); sodium hyrdoxide (Cat # 221465); ascorbic acid (Cat # A4544); hydroxylamine solution (Cat # 467804). The following reagents were sourced from Thermo Fisher: TrypLE™ Express Enzyme (Cat #12604013) (Gibco, New York, NY, USA); Pierce™ BCA Protein Assay Kit (Cat #23225) (Thermo Scientific, Rockford, IL, USA). The UCP-5 peptide was synthesised by the Payne Laboratory with sequence: EEGVLALYSGIAPALLR.
4
Botulinum Neurotoxin Proteolytic Assay
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Botulinum neurotoxin is highly toxic and requires appropriate safety measures. All neurotoxins were handled in a class 2 biosafety cabinet equipped with HEPA filters. Commercially purified BoNT/G complex toxin was purchased (Metabiologics, Madison, WI). Sequencing-grade modified trypsin at 0.5 mg/mL in 50 mM acetic acid and sequencing grade chymotrypsin at 1 μg/μL in 50 mM ammonium bicarbonate was purchased (Roche, Pleasanton, CA). All chemicals were from Sigma-Aldrich (St. Louis, MO) except where indicated.
5
Cryo-pulverization and Tissue Lysis for Proteomics
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Frozen liver tissue samples were transferred into prechilled tubes and cryo-pulverized in a CP02 Automated Dry Pulverizer (Covaris, Woburn, MA, USA) using an impact level of 3 according to the manufacturer's instructions. Powdered tissue was lysed in 8 M urea/0.5 M ammonium bicarbonate (Roche Diagnostics, Mannheim, Germany) by ultrasonication (18 cycles of 10 s) using a Sonopuls HD3200 (Bandelin, Berlin, Germany). Pierce 660 nm Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA) was used for protein quantification. 20 μL of lysate containing 20 μg of protein were processed for digestion. Disulfide bonds were reduced with 45 mM dithiothreitol/20 mM tris(2-carboxyethyl) phosphine (30 min, 56 °C). Reduced cysteine side chains were alkylated by adding 100 mM iodoacetamide (30 min, room temperature), followed by quenching the remaining iodoacetamide with dithiothreitol (90 mM, 15 min, room temperature). Sequential 2-step digestion was performed, firstly with Lys-C (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) for 4 h (1:50 enzyme to protein ratio) and subsequently with modified porcine trypsin (Promega, Madison, WI, USA) for 16 h at 37 °C (1:50 enzyme to protein ratio). After digestion, samples were dried before analysis using a vacuum centrifuge.
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