CSF samples were dissolved in Laemmli buffer without 2-mercaptoethanol, boiled for 3 min and loaded on SDS-polyacrylamide gels (194–1502, FUJIFILM Wako) [33 (link)]. After SDS-PAGE, protein bands were visualized with a Silver Stain II kit (FUJIFILM Wako). For immunoblotting, proteins separated on gels were transferred to nitrocellulose membranes. The membranes were blocked in 3% skim milk, incubated sequentially with anti-transferrin antibody (Bethyl Laboratories, Montgomery, TX, USA) and horseradish peroxidase-labeled anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and developed using a SuperSignal West Dura Chemiluminescence Substrate Kit (Pierce Biotechnology, Rockford, IL, USA). For lectin blotting, membranes were blocked in 1% BSA and incubated with a mannose-binding lectin, rBC2L-A (026-18691, FUJIFILM Wako). Prior to use, rBC2L-A lectin was biotinylated using Ez-Link NHS-Biotin (#20217, Thermo Fisher Scientific, Waltham, MA, USA). Membranes were further incubated with horseradish peroxidase-labeled streptavidin (SA00001-0, Cosmo Bio Co., Tokyo, Japan), and developed. Biotinylated Urtica dioica Lectin (UDA) (BA-8005-1, Cosmo Bio Co.) and biotinylated Concanavalin A (ConA) (BA-1104-5, Cosmo Bio Co.) were also used for mannose-binding lectins.
Supersignal west dura chemiluminescence substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperSignal West Dura Chemiluminescence Substrate Kit is a reagent used to detect and quantify proteins in Western blot analysis. The kit provides a chemiluminescent substrate that generates a signal when exposed to the enzyme-labeled detection antibody, allowing for the visualization and quantification of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Silver stain 2 kit, by Fujifilm (3 mentions)
Anti tf antibody, by Fortis Life Sciences (2 mentions)
Ez link nhs biotin, by Thermo Fisher Scientific (1 mentions)
Sds polyacrylamide gel, by Fujifilm (1 mentions)
Supersep ace, by Fujifilm (1 mentions)
Epr12763, by Abcam (1 mentions)
Hrp labeled anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
4 protocols using supersignal west dura chemiluminescence substrate kit
1
Glycoprotein Analysis in CSF Samples
2
Quantification of Transferrin Isoforms in CSF
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We quantified Tf isoforms, brain-type and serum-type (formerly Tf-1 and Tf-2, respectively) by immunoblotting as previously described11) (link). Briefly, each CSF sample was dissolved in sample buffer and boiled for 5 min. The CSF sample was loaded onto 7.5% SDS-polyacrylamide gels (SuperSepTM Ace; Wako Pure Chemical Industries, Osaka, Japan) and transferred to nitrocellulose membranes. After blocking the membrane with 3% skim milk in phosphate-buffered saline with Tween (PBST), and incubating sequentially with an anti-Tf antibody (Bethyl Laboratories) and an HRP-labeled anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), the protein was detected using a SuperSignal West Dura Chemiluminescence Substrate Kit (Pierce Biotechnology, Rockford, IL). Signal intensities were quantified by chromato-scanning with a CS Analyzer 2.0 (ATTO, Tokyo, Japan).
3
Western Blot Analysis of Brain Proteins
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Brain tissue was washed three times with PBS containing 1% protease inhibitor cocktail. Protein in the tissue was extracted with PBS containing 1% TritonX-100 and 1% protease inhibitor cocktail. The extracts were dissolved in Laemmli sample buffer, boiled for 3 min, and loaded on a gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (SuperSep Ace 7.5%, FUJIFILM Wako, Osaka, Japan). After SDS-PAGE, protein bands were visualized with a Silver Stain II kit (FUJIFILM Wako). For immunoblotting, proteins separated on gels were transferred to nitrocellulose membranes. The membranes were blocked in 3% skim milk, and incubated anti-transferrin antibodies labeled with HRP (A90-129P, anti-Mouse Transferrin; A80-128P, anti-Human Transferrin, Bethyl Laboratories, Montgomery, TX, USA). Anti-NeuN antibody was used for detecting the antigen (EPR12763, Abcam, Cambridge, UK). The membranes were developed using a SuperSignal West Dura Chemiluminescence Substrate Kit (Pierce Biotechnology, Rockford, IL, USA).
4
Protein Analysis of CSF and Serum
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CSF and serum samples were dissolved in Laemmli buffer without 2-mercaptoethanol, boiled for 3 min, and loaded on SDS-polyacrylamide gels (194–1502, FUJIFILM Wako) [27 (link)]. After SDS-PAGE, protein bands were visualized with a Silver Stain II kit (FUJIFILM Wako). For immunoblotting, proteins separated on gels were transferred to nitrocellulose membranes. The membranes were blocked in 3% skim milk, and incubated sequentially with the following combinations of primary and secondary antibodies: anti-Tf antibody (Bethyl Laboratories, Montgomery, TX, USA) and horseradish peroxidase (HRP)-labeled anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA); anti-PDGS antibody (PA1-46023, Thermo Fisher Scientific, Waltham, MA, USA) and HRP-labeled anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA); anti-TTR antibody (ab9015, Abcam, Cambridge, CB2 0AX, UK) and HRP-labeled anti-sheep IgG antibody (#31480, BiotechnologyThermo Fisher Scientific). The protein bands were visualized with a SuperSignal West Dura Chemiluminescence Substrate Kit (Pierce Biotechnology, Rockford, IL, USA). Purified standards of GlcNAc and Man-Tf mixture were purified from CSF as described previously [15 (link)]. His-tagged TTR (89-7754-27) and L-PGDS (E-PKSH030653.10) were purchased from Elabscience Biotechnology Inc., (Houston, TX, USA) and Biomol (Hamburg, Germany)
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