Iodoacetamide (IAA), glutathione (GSH), DL-dithiothreitol (DTT), tris(2-chloroethyl) phosphate (TCEP), copper sulfate (CuSO4), S-methyl methanethiosulfonate (MMTS) and calcium chloride (CaCl2) were purchased from Sigma-Aldrich unless otherwise specified. Fmoc-Cys and N-ethylmaleimide (NEM) was obtained from Heowns and J&K Scientific, respectively. Biotin-PEG3-azide, DADPS-Biotin-Azide and click chemistry auxiliary reagents (TBTA and BTTAA) was purchased from Click chemistry tools. Cy3-Azide and desthioBiotin-PEG3-azide was obtained from Okeanos and Confluore Biotechnology, respectively. Four peptides containing one cysteine, GCSWDYKN was synthesized from GL Biochem and the others were purchased from SciLight Biotechnology. All cell culture related reagents, such as fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM) and penicillin-streptomycin were purchased from Biological Industries (BI). Three kinds of proteins containing one free cysteine residues, β-lactoglobulin A, bovine serum albumins and papain were obtained from Sigma-Aldrich. All antibodies used for immunoblotting were purchased from Abcam except HRP-labeled Streptavidin (Beyotime Biotechnology), and other chemical or biological reagents were obtained from commercial suppliers without any manipulation.
β lactoglobulin a
Manufactured by Merck Group
Sourced in United Kingdom
β-lactoglobulin A is a protein found in whey, a byproduct of cheese production. It is commonly used as a standard and reference material in various laboratory applications, such as protein analysis and characterization.
Automatically generated - may contain errors
Lab products found in correlation
Yeast alcohol dehydrogenase, by Merck Group (2 mentions)
Synapt hdms mass spectrometer, by Waters Corporation (2 mentions)
Masslynx v4, by Waters Corporation (2 mentions)
Driftscope 2, by Waters Corporation (2 mentions)
Massign, by Waters Corporation (2 mentions)
Concanavilin a, by Merck Group (2 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Transthyretin, by Merck Group (2 mentions)
Concanavalin a (cona), by Merck Group (2 mentions)
Bovine serum albumins, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Dulbecco s modified eagle s medium dmem, by Sartorius (1 mentions)
Penicillin streptomycin, by Sartorius (1 mentions)
Biotin peg3 azide, by Vector Laboratories (1 mentions)
Dadps biotin azide, by Vector Laboratories (1 mentions)
Hrp labeled streptavidin, by Beyotime (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Papain, by Merck Group (1 mentions)
Trypsinogen, by Merck Group (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
7 protocols using β lactoglobulin a
1
Comprehensive Cysteine Labeling Protocol
2
Protein Assay Standards Characterization
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The following specimens were purchased from Sigma-Aldrich (St. Louis, MO, USA): (1) human serum albumin (fatty acid and human globulin free (~99%)), (2) albumin from bovine serum, (3) human γ-globulin, (4) β-lactoglobulin A from bovine milk (>90%), (5) β-lactoglobulin B from bovine milk (>90%), (6) lysozyme from chicken egg white, (7) trypsinogen from bovine pancreas, (8) ovalbumin, and (9) pyridine-N-oxide (PyO) with a purity of 95% for the NMR measurements and analysis.
3
Characterization of Skp-OMP Complexes by IMS-MS
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Skp:OMP complexes were prepared by rapid dilution of the denatured OMP (400 μM in 8 M urea, 50 mM glycine-NaOH, pH 9.5) to a final concentration of 5 μM into a solution of Skp (5 μM in 50 mM glycine-NaOH, pH 9.5). The samples were then buffer exchanged into 200 mM ammonium acetate, pH 10 using Zeba spin desalting columns (Thermo Scientific, UK) immediately prior to MS analysis. nanoESI-IMS-MS spectra were acquired using a Synapt HDMS mass spectrometer (Waters Corporation, UK) using platinum/gold-plated borosilicate capillaries prepared in-house. Typical instrument parameters include: capillary voltage 1.2-1.6 kV, cone voltage 40 V, trap collision voltage 6 V, transfer collision voltage 10 V, trap DC bias 20 V, backing pressure 4.5 mBar, IMS gas pressure 0.5 mBar, travelling wave height 7 V, travelling wave velocity 250 ms-1. Data were processed using MassLynx v4.1, Driftscope 2.5 (Waters Corporation, UK) and Massign55 (link). CCSs were estimated by a calibration approach28 (link),33 (link),56 using arrival time data for ions with known CCSs (β-lactoglobulin A, avidin, concanavilin A and yeast alcohol dehydrogenase, all Sigma Aldrich, UK). Estimated modal CCSs are shown as mean ± standard deviation of three independent experiments. Theoretical CCSs for globular proteins with a given effective gas phase density were calculated according to published methods57 (link).
4
IM-MS Analysis of Purified Aurora A
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IM-MS analysis was performed on a Waters Synapt G2-Si instrument. Purified Aurora A was buffer exchanged into 50 mM NH4OAc (LC grade, Sigma) as previously described [69 (link)]. Typically, 1–3 μl of 2–5 μM sample was analysed using borosilicate emitters (Thermo ES 387). Spraying voltage was adjusted to 1.1–1.8 kV, sampling cone was 20 V. Pressure in the travelling wave (T-wave) ion mobility cell was 2.78 mbar (nitrogen), wave height was kept at 30 V, wave velocity at 750 m/s. In order to experimentally determine collision cross section (CCS), drift time through the T-wave mobility cell was performed using β-lactoglobulin A (Sigma L7880), avidin (Sigma A9275), transthyretin (Sigma P1742), concanavalin A (Sigma C2010) and serum albumin (Sigma P7656) according to standard protocols. The exact hard sphere scattering (EHSS) model implemented in the Mobcal software was used to calculate CCS values on the basis of X-ray structures, as described previously [69 (link)].
5
Protein Thiol Labeling and Quantification
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PhNHOH, NOB, bovine Hb, human Hb, β-lactoglobulin A from bovine milk (β-LGB-A), iodoacetamide (IAM), DTT, GSH, GSSG, and sequencing grade trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich (St. Louis, MO). LysC/trypsin mix was obtained from Promega Corporation (Madison, WI). The model synthetic peptides AcPAAKAA, AcPAACAA and AcPAAHAA were custom synthesized by Biomatik, Inc. (Wilmington, DE). Human acetyl ACTH (1–17) and acetyl γ-endorphin peptides were obtained from American Peptide Company (Vista, CA). ELHCDKL peptide was synthesized in-house using Fmoc-Leu-Wang resin with amino acids obtained from AnaSpec Inc. (San Jose, CA). Amicon Ultra Centrifugal Filters, 0.5 mL/10 kDa and C18 Zip-Tips were from Millipore Ltd. (Bedford, MA). Pooled human whole blood from random, deidentified donors was obtained from a commercial supplier (Bioreclamation, LLC; Westbury, NY). All solvents were high purity grade from Fisher Scientific Co. (Pittsburgh, PA).
6
Characterization of Skp-OMP Complexes by IMS-MS
7
Determination of Protein CCS by IM-MS
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To experimentally determine CCS, the measured drift time through the T-wave mobility cell of β-lactoglobulin A (Sigma L7880), avidin (Sigma A9275), transthyretin (Sigma P1742), concanavalin A (Sigma C2010) and serum albumin (Sigma P7656) was calculated according to standard protocols [54 (link)]. The exact hard sphere scattering (EHSS) model implemented in the Mobcal software was used to calculate CCS values on the basis of X-ray structures [55 (link)].
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