Serum samples were collected and cryopreserved from each patient at baseline, 6 months, 12 months and 24 months. Samples were not available from all patients at each time-point; a total of 475 samples were available for SELDI-TOF mass spectrometry. Samples were randomized, and staff were blinded to the treatment arms. CM10 ProteinChip arrays (Bio-Rad Laboratories) were primed with binding buffer (50 mM ammonium acetate, 0.01% Triton X-100, pH 4·0) and incubated at room temperature (RT) for 5 min. A 1:10 dilution of serum in binding buffer was then applied to the array and incubated at RT for 1 hr. The arrays were washed twice with binding buffer and deionized water. Saturated sinapinic acid (0.7 µL) was applied twice to each spot on the arrays. Time-of-flight spectra were generated using a PCS-4000 mass spectrometer (Bio-Rad). Low-range spectra (mass/charge (m/z) ratio 0 – 20,000) were obtained at a laser energy of 3000 nJ, with a focus mass of 6000 and the matrix attenuated to 1000. High-range spectra (m/z 10,000 – 75,000) were obtained at a laser energy of 3900 nJ, with a focus mass of 30,000 and the matrix attenuated to 10,000. Mass accuracy was calibrated externally using All-in-One Peptide or Protein molecular mass standards (Bio-Rad).
Protein molecular mass standards
Manufactured by Bio-Rad
Protein molecular mass standards are laboratory reagents used to determine the molecular weights of unknown protein samples. They consist of a mixture of pre-stained proteins with known molecular masses that can be used as reference markers when analyzing proteins using techniques such as SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis).
Automatically generated - may contain errors
Lab products found in correlation
Western lightning plus ecl chemiluminescence kit, by PerkinElmer (3 mentions)
Cm10 proteinchip array, by Bio-Rad (2 mentions)
Pcs 4000 mass spectrometer, by Bio-Rad (2 mentions)
All in one peptide, by Bio-Rad (2 mentions)
Vldlr af2258, by R&D Systems (2 mentions)
Immobilon polyvinylidene difluoride membrane, by Merck Group (2 mentions)
γ globulin, by Bio-Rad (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Thyroglobulin, by Bio-Rad (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Gapdh mab374, by Merck Group (1 mentions)
Pparγ sc 7273, by Santa Cruz Biotechnology (1 mentions)
Cd36 nb400 144, by Novus Biologicals (1 mentions)
Actin a5441, by Merck Group (1 mentions)
Nrf2 sc 722, by Santa Cruz Biotechnology (1 mentions)
Atf4 sc 200, by Santa Cruz Biotechnology (1 mentions)
Vldlr, by Santa Cruz Biotechnology (1 mentions)
Nqo1 sc 393736, by Santa Cruz Biotechnology (1 mentions)
Eif2α, by R&D Systems (1 mentions)
7 protocols using protein molecular mass standards
1
SELDI-TOF Mass Spectrometry of Serum Biomarkers
2
Oligomerization States of EcAhpC Proteins
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To determine the oligomerization state of oxidized EcAhpC and EcAhpC1–172, 10 µM of proteins in 50 mM phosphate (pH 7.0) were incubated at 25 °C, 48 °C, 50 °C or 53 °C for 60 min. After cooling to room temperature, the samples were subjected to high speed centrifugation and loaded on a SEC column (Superdex 200 10/300 GL column (GE Healthcare)), equilibrated with 50 mM Tris, pH 7. The samples were detected by absorbance at 280 nm. For the reduced EcAhpC, oxidized WT EcAhpC was reduced with 20 mM DTT in 50 mM phosphate buffer (pH 7.0) for 1 h. Reduced proteins were separated from excess of DTT using a PD-10 desalting column (GE Healthcare). This pre-reduced EcAhpC was incubated at 25 °C or 53 °C for 60 min in a 50 mM phosphate (pH 7.0) containing 1 mM TCEP and subjected to SEC analysis.
Protein molecular mass standards (Bio-rad), thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa) were used to calibrate the SEC column. The column void volume was determined with blue dextran 2000 (Amersham Pharmacia Biotech Co., Little Chalfont, England).
Protein molecular mass standards (Bio-rad), thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa) were used to calibrate the SEC column. The column void volume was determined with blue dextran 2000 (Amersham Pharmacia Biotech Co., Little Chalfont, England).
3
Protein Extraction and Western Blot Analysis
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Isolation of total and nuclear extracts was performed as described elsewhere [21 (link)]. Proteins (30 μg) were separated by SDS-PAGE on 10% (w/v) acrylamide separation gels and transferred to Immobilon polyvinylidene difluoride membranes (Merck Millipore). Western blot analysis was performed using antibodies against HIF1α (sc-10,790), LIPIN1 (sc-98,450), Histone H3 (sc-10,809), SREBP1 (sc-365,513), Nrf2 (sc-722), NQO1 (sc-393,736), PPARγ (sc-7273) (Santa Cruz Biotechnology), SIRT3 (#5490), phospho-mTOR Ser2481 (#2974), mTOR (#2972), Keap1 (#4678 s) (Cell Signaling Technology Inc., Danvers, MA), GAPDH (MAB374) (Merck Millipore), CD36 (NB400–144) (Novus Biologicals, Centennial, CO), VLDLR (AF2258) (R&D Systems, Minneapolis, MN). Detection was performed with the Western Lightning™® Plus-ECL chemiluminescence kit (PerkinElmer, Waltham, MA, USA). The size of detected proteins was estimated using protein molecular-mass standards (Bio-Rad, Hercules, CA).
4
Western Blot Analysis of Cellular Protein Extracts
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Isolation of total and nuclear extracts was performed as described elsewhere [28] (link). Proteins (30 μg) were separated by SDS-PAGE on 10% acrylamide separation gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore). Western blot analysis was performed using antibodies against VLDLR (sc-18824), Nrf2 (sc-722), Nqo1 (sc-393736), ATF4 (sc-200) (Santa Cruz), VLDLR (AF2258) (R&D system), eIF2α (9722), phospho-eIF2α (Ser51) (9721), IgG control (2729S) (Cell Signaling Technology Inc., Danvers, MA), actin (A5441) (Sigma–Aldrich, Madrid, Spain). Detection was achieved using the Western Lightning® Plus-ECL chemiluminescence kit (PerkinElmer, Waltham, MA, USA). The equal loading of proteins was assessed by Ponceau S staining. The size of detected proteins was estimated using protein molecular-mass standards (Bio-Rad).
5
Comparative SDS-PAGE Analysis of PSA Calibrants
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Two different lots of the Calbiochem PSA (D10010682 (lot a) and D00116361 (lot b)) CS, as well as, the Scripps and Fitzgerald PSA CS were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R&D assay PSA calibrant (R&D, #1344-SE), prepared according to manufacturer recommendations, was also examined (Fig. 3 ). SDS-PAGE was performed according to Laemmli13 (link) with the below modifications. Samples (3 μg) were diluted in a 1 to 1 ratio with buffer (Bio-Rad Laboratories, #161–0737) under reducing conditions with 5% β-mercaptoethanol. PSA samples and 10 μL of protein molecular mass standards (Bio-Rad, #161–0137) were boiled for 5 minutes. After heating, samples were loaded on a 1.0 mm thick, 12.5%T/3.3%C polyacrylamide gel. Gels were run in a Mini Protean Tetra Electrophoresis Cell (Bio-Rad, #165–0827) and a voltage of 80 V for 15 min followed by 80 min at 120 V was applied. Proteins were silver stained and destained (Thermo, #24612) according to manufacturer recommendations.
6
SELDI-TOF Mass Spectrometry Protocol
7
Western Blot Analysis of Key Cellular Markers
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Isolation of total and nuclear extracts was performed as described elsewhere [28] . Western blot analysis was performed using antibodies against total and phospho-Akt (Ser 473 ), adenylate cyclase, BACE1, CHOP, total and phospho-CREB (Ser 133 ), total and phospho-eIF2, GRP78/BiP, insulin receptor -subunit (IRNRF1, total and phospho-PKA (Thr 197 ), total and p-STAT3 (Tyr 705 )(Cell Signaling Technology Inc., Danvers, MA), OXPHOS (Mito Sciences, Eugene, OR), A42 (Biolegend), sAPP (Covance, Alnwick, UK), PGC-1Abcam, Cambridge, United Kingdom), GAPDH, IBlamin B, Oct-1, p65, PPAR, PPAR/, prohibitin (Santa Cruz), total and phospho-IRS-1 (Ser 307 ) (Millipore, Billerica, MA) and -actin (Sigma, St. Louis, MO). Detection was achieved using the Western Lightning® Plus-ECL chemiluminescence kit (PerkinElmer, Waltham, MA). The equal loading of proteins was assessed by Ponceau S staining. The size of detected proteins was estimated using protein molecular-mass standards (Bio-Rad, Hercules, CA). For validation, we used a protein marker (Precision Plus Protein Dual Color Standards 1610374; Bio-Rad, Hercules, CA, USA), on the same blots. All of these commercially available antibodies showed a single distinct band at the molecular weight indicated in the datasheets.
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