To analyse PHB2 phosphorylation, phos-tag SDS–PAGE was performed using precast SuperSep gels (50 μM phos-tag acrylamide and 100 μM ZnCl2, Wako Chemicals, 190–16721), as previously described with some modification45 (link). Molecular size markers for phos-tag SDS–PAGE used the WIDE-VIEW Prestained Protein Size Marker III (Wako Chemicals, 236–02463). The percentage of phosphorylation was calculated according to the formula: (phosphorylated PHB2 band area/total PHB2 band area) × 100.
Supersep gel
Manufactured by Fujifilm
Sourced in Japan
SuperSep gel is a laboratory equipment product designed for protein separation and analysis. It is a pre-cast polyacrylamide gel that is used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins based on their molecular weight. The gel is available in various formats and concentrations to accommodate different protein analysis needs.
Automatically generated - may contain errors
Lab products found in correlation
Zncl2, by Fujifilm (1 mentions)
Wide view prestained protein size marker 3, by Fujifilm (1 mentions)
Complete mini, by Roche (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Anti c myc antibody, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg antibody, by Promega (1 mentions)
Anti cyp b, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated anti mouse igg antibody, by Promega (1 mentions)
Anti pin1 antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Anti gapdh ab, by Abcam (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Wet transfer apparatus, by Bio-Rad (1 mentions)
Autoflex speed, by Bruker (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Mascot server, by Matrix Science (1 mentions)
5 protocols using supersep gel
1
Phosphorylation Analysis of PHB2 Using Phos-tag SDS-PAGE
2
Western Blot Analysis of Coronavirus Infection
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The cell membranes were disrupted with cell lysis buffer [10 mM Tris–HCl, pH 7.8, 1 mM ethylenediamine tetraacetic acid (EDTA), 1% NP-40, and 0.15 M NaCl], including Complete Mini (Roche Diagnostics, Tokyo, Japan) at 20 h after infection. The cell lysates were resolved by electrophoresis on 12.5% SuperSep gels (WAKO, Tokyo, Japan) and Western blotted on to Immobilon-P membranes (Millipore, Tokyo, Japan). Non-specific protein binding was blocked with 5% non-fat dry milk, and then the membranes were incubated with the primary antibodies [anti-FCoV nucleocapsid (N) antibody (FIPV3-70; MyBioSource, CA, USA), anti-c-Myc antibody (Santa Cruz Biotechnology, CA, USA), anti-Pin1 antibody (Cell Signaling Technology, Tokyo, Japan), anti-Cyp B (Thermo Fisher Scientific, Yokohama, Japan), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Calbiochem, CA, USA)] for 1 h. Antigen signals were visualized by reacting proteins on the membranes with horseradish peroxidase-conjugated anti-mouse IgG antibody (Promega) and/or anti-rabbit IgG antibody (Promega) followed by an enhanced chemiluminescence substrate (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific) according to the manufacturer's protocol.
3
Western Blot Protein Detection
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Total cell protein was extracted using RIPA buffer. Proteins in the lysate were resolved by SDS-PAGE using a 5-20% SuperSep gel (Wako, Japan). The resolved proteins were transferred to nitrocellulose membrane. Protein bands were incubated with primary antibody overnight at 4°C. Signals were visualized by enhanced chemiluminescence according to the manufacturer's instructions (GE Healthcare). Anti-RPN2 Ab was from AVIVA SYSTEMS and anti-GAPDH Ab was from Abcam.
4
Western Blot Analysis of PROK2 Protein
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HCT116 and DLD-1 cells were placed into 6-well plates (5 × 104 cells/well) with RPMI containing 10% FBS and incubated at 37°C in 5% CO2. Total cell protein was extracted using RIPA lysis buffer supplemented with protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and sodium orthovanadate). Denatured proteins were separated by SDS-PAGE using 15% SuperSep Gel (Wako, Japan). The resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using the wet-transfer apparatus (Bio-Rad, Hercules, CA, USA) [45 ]. Following electrophoretic transfer, the protein bands were blocked in 5% skim milk overnight at 4°C and incubated with anti-PROK2 antibody (Novus Biochemicals). The blots were washed thrice in T-TBS and incubated with horseradish peroxidase (HRP)-tagged goat anti-rabbit IgG secondary antibodies for 1 h at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence according to the manufacturer’s instructions (Amersham, Piscataway, NJ, USA). The experiment was performed in triplicate.
5
Polypeptide Separation and Mass Spectrometry Analysis
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Polypeptides denatured with dithiothreitol were separated on a SuperSep gel containing 10–20% acrylamide (Wako, Tokyo, Japan) and then stained with Coomassie Blue (Supplementary Fig. 2c ). A gel imaging system (ChemiDoc XRS + system; Bio-Rad, Hercules, CA, USA) was used to photograph the stained gels. Separated polypeptides (a band containing ApcC and CpcD) were identified by MS. The obtained protein bands were treated by in situ digestion using trypsin69 (link). The fragmented peptides were analyzed by peptide mass fingerprinting and MS/MS using Autoflex speed (Bruker Daltonik GmbH, Bremen, Germany). The obtained mass spectra were analyzed by using the MASCOT server (Matrix Science Inc., Boston, MA, USA).
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