Fractogel DEAE‐650M, HW‐50 and HW‐55 gel filtration resins were purchased from Merck (Darmstadt, Germany). Toyopearl Phenyl‐650M was purchased from Toyo Soda Manufacturing (Tokyo, Japan). Ultragel‐HA was acquired from IBF Biotechnics (Pairs, France). Protein assay dye and SDS/PAGE molecular weight standards were obtained from Bio‐Rad (Hercules, CA, USA). Sephacryl S‐200 HR16/60 fast performance liquid chromatography (FPLC) column was obtained from GE Healthcare Bio‐Science (Uppsala, Sweden). FPLC molecular weight standards, 4‐aminoantipyrine (4AA), peroxidase and all the sugars were from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytic reagent grade.
Sds page molecular weight standards
Manufactured by Bio-Rad
Sourced in United States
SDS-PAGE molecular weight standards are a set of pre-stained proteins with known molecular weights. They are used as reference markers to estimate the molecular weights of unknown protein samples during SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
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Lab products found in correlation
Taqman snp genotyping assay and reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Chemiluminescence kit, by Cytiva (3 mentions)
Anti β tubulin, by Abcam (2 mentions)
Protein assay dye, by Bio-Rad (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Anti vdr, by Elabscience (1 mentions)
Coomassie brilliant blue r 250, by Nacalai Tesque (1 mentions)
Anti n smase, by Abcam (1 mentions)
Anti caveolin 1, by Santa Cruz Biotechnology (1 mentions)
Fluorescein isothiocyanate fitc conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
Quetol 812, by Nisshin EM (1 mentions)
Propylene oxide, by Nisshin EM (1 mentions)
Anti caspase 1, by Abcam (1 mentions)
Anti asmase, by Abcam (1 mentions)
E coli bl21 de3 cells, by New England Biolabs (1 mentions)
9 protocols using sds page molecular weight standards
1
Chromatography Techniques for Protein Purification
2
Quantification of Molecular Markers in Cells
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Anti-GFAP and anti-SOX2 antibodies were obtained from Dako, Agilent (Santa Clara, CA, United States), anti- VDR and anti-aSMase were from Elabscience (Houston, TX, United States) and anti-β tubulin was from Sigma Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TaqMan SNP Genotyping Assay and Reverse Transcription kit were purchased from Applied Biosystems (Foster City, CA, USA). RNAqueous®-4PCR kit was from Ambion Inc. (Austin, Texas). SDS-PAGE molecular weight standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Chemiluminescence kit was purchased from Amersham (Rainham, Essex, UK).
3
Chalaza Protein Extraction and Characterization
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After the separation of chalaza, it was carefully rinsed three times by ringer solution to remove any substances that come from the egg white. The chalaza was then centrifugated and freeze-dried into powders. We used 0.3 wt% chalaza suspensions for the SDS-Page. Chalaza materials were separated by 7.5% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis;) or 3–10% gradient gel “PAGEL” (Atto, Tokyo, Japan). The gels were stained with 0.05% Coomassie Brilliant Blue R-250 (CBB; Nacalai Tesque, Kyoto, Japan). The molecular weight was estimated from a calibration curve obtained by SDS-PAGE molecular weight standards (Bio-Rad, Hercules, CA, USA) or Hi-Mark pre-stained high-molecular protein standards (Invitrogen, Carlsbad, CA, USA).
4
Recombinant Manganese Superoxide Dismutase Radioprotection
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The rMnSOD radioprotective protein, discovered and obtained in the recombinant form by A.Mancini, was provided by the Molecular Biology and Viral Oncology Unit, Department of Experimental Oncology, Istituto Nazionale Tumori Fondazione G. Pascale—IRCCS, Naples, Italy [15 (link)]. Sterile 2 μg aliquots of rMnSOD in 0.5 mL of sterile saline phosphate buffer (PBS) were prepared and stored at −80 °C. Anti-nSMase, and anti-βtubulin were from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were from Santa Cruz. Anti-rMnSOD was from InBios International (Washington, WA, USA). TaqMan SNP Genotyping Assay and Reverse Transcription kit were purchased from Applied Biosystems (Foster City, CA, USA). RNAqueous®-4PCR kit was from Ambion Inc. (Austin, TX, USA). SDS-PAGE molecular weight standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Chemiluminescence kits were purchased fromAmersham (Rainham, Essex, UK).
5
Lipid Profiling of Thyroid Cells
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Anti- Signal transducer and activator of transcription-3 (STAT3), anti-TSHR, anti-Caveolin 1, fluorescein isothiocyanate (FITC)-conjugated secondary antibody were obtained from Santa Cruz Biotechnology, Inc. (California, USA); SDS-PAGE molecular weight standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Chemiluminescence kits was purchased from Amersham (Rainham, Essex, UK). Cholesterol (CHO), TSH and cAMP EIA kits were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) and CABRU SAS (Milan, Italy), respectively. Sphingomyelin (SM) 18∶1 12∶0, SM 18∶1 16∶0, SM 18∶1 18∶1, SM 24∶0, phosphatidylcholine (PC) 16∶0 18∶1, PC 16∶0 20∶4, PC 18∶1 18∶0, ceramide 18∶1 16∶0, ceramide 18∶1 18∶0, ceramide 18∶1 20∶0, ceramide 18∶1 24∶0 were purchased from Avanti (Avanti Polar, Alabaster, USA).
6
Immunofluorescence and Electron Microscopy
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IPG strips and buffers were purchased from GE Healthcare (Buckinghamshire, England). Ammonium bicarbonate and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin was purchased from Promega (Madison, WI, USA). HCCA and peptide calibration standards were purchased from Bruker Daltonics (Billerica, MA, USA). SDS-PAGE molecular weight standards were purchased from Bio-Rad (Hercules, CA, USA). Anti-acetylated α -tubulin antibody (clone 6-11B-1) was purchased from Funakoshi (Tokyo, Japan). Alexa 488-conjugated anti-mouse antibody was purchased from Invitrogen (Carlsbad, CA, USA). OsO4, propylene oxide and Quetol 812 were purchased from Nisshin EM Co. (Tokyo, Japan). All the other reagents were purchased from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).
7
Purification of a Protein via Anion Exchange
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The EP was centrifuged at 2,270xg for 15 min; the obtained supernatant was dialyzed at room temperature, using a cellulose membrane with a pore size of 6–8kDa, against 50mM MOPS buffer, pH 7 (3-N-morpholino propanol sulfonic acid), including 0.1M NaCl; this for 24 h with at least three shock absorber replacements, and gentle agitation. The dialyzed sample was centrifuged again at 2,270xg for 15 min; and the supernatant obtained was concentrated to 2.6 mg/mL using an ultrafiltration system (AMICON), with a 10kDa membrane. Subsequently, it was filtered using a membrane with a pore size of 0.22μm and passed, at a flow of 1 mL/min, through a column with anion exchange matrix (Aligent BioWax NP 5, 4.6 × 250mm) previously equilibrated with MOPS-NaCl buffer, in a high resolution system. The fractions were eluted using a linear gradient from 0.1 to 1M NaCl in 50mM MOPS buffer, pH 7. The fractions purity was analyzed by 15% SDS-PAGE and the molecular weight marker was used as a reference for migration. 200 kDa at 6.5kDa (SDS-PAGE Molecular weight Standards BioRad®, Broad Range No. 161-0317). The development of the protein bands was carried out by silver nitrate staining [15] (link).
8
Molecular Mechanisms of Oxidative Stress
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The rMnSOD protein was provided by the Molecular Biology and Viral Oncology Unit, Department of Experimental Oncology, “Istituto Nazionale Tumori Fondazione G. Pascale”—IRCCS, Naples, Italy [34 (link)]. Anti-aSMase, anti-nSMase1, anti-Caspase1, and anti-β-tubulin were obtained from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were obtained from Santa Cruz. The TaqMan SNP Genotyping Assay and Reverse Transcription kit were obtained from Applied Biosystems (Foster City, CA, USA). The RNAqueous®-4PCR kit was obtained from Ambion Inc. (Austin, Texas, USA). The SDS-PAGE molecular weight standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Chemiluminescence kits were purchased from Amersham (Rainham, Essex, UK).
9
Recombinant Protein Expression in E. coli
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