For activation of globupain and substitution variants (Table 1 ), a purified enzyme sample (< 5 mg/mL) was incubated at 75°C for up to 4.5 h in 20 mM tri-sodium citrate dihydrate, 150 mM NaCl, pH 5.5 (at room temperature) with 2.5 mM DTT and 1 mM CaCl2, respectively (activation buffer). To investigate if globupain could in-trans activate, 10 μg of activated WT globupain was mixed with 10 μg of inactive C185A variant. The number and size of cleavage products were assessed by visualization of protein bands on 8–16% SurePAGE precast gels (GenScript) using MES SDS running buffer (GenScript) in a Bio-Rad Mini-PROTEAN Tetra Cell (BioRad, Hercules, CA, USA). For sample preparation, 4´ lithium dodecyl sulfate (LDS) sample buffer (GenScript) with 2-mercaptoethanol was mixed with the protein sample, followed by denaturing at 95°C for 10 min. Gels were stained with InstantBlue™ ultrafast protein stain (Abcam, Cambridge, United Kingdom), and the size of bands was indicated by broad multi-color pre-stained protein standard (GenScript). Edman sequencing was performed on a Shimadzu PPSQ-53A at the Iowa State University Protein Facility, USA.
Surepage precast gel
Manufactured by GenScript
Sourced in United States, China
SurePAGE™ Precast Gels are a line of pre-cast polyacrylamide gels designed for protein electrophoresis. They are available in various percentages and formats to accommodate different applications.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tetra cell, by Bio-Rad (2 mentions)
Mes sds running buffer, by GenScript (2 mentions)
Instantblue ultrafast protein stain, by Abcam (2 mentions)
Ppsq 53a, by Shimadzu (2 mentions)
Broad multi color pre stained protein standard, by GenScript (2 mentions)
Ab228592, by Abcam (1 mentions)
Ab150422, by Abcam (1 mentions)
Ab32559, by Abcam (1 mentions)
Ab179483, by Abcam (1 mentions)
Nb100 664, by Novus Biologicals (1 mentions)
M per lysis buffer, by Thermo Fisher Scientific (1 mentions)
P erk and erk, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
O glcnac, by Thermo Fisher Scientific (1 mentions)
β actin, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Pol ι, by Proteintech (1 mentions)
Hrp conjugated anti mouse or anti rabbit secondary antibody, by MultiSciences Biotech (1 mentions)
8 protocols using surepage precast gel
1
Activation of Globupain Variants
2
Activation and Cleavage Analysis of Globupain
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For activation of globupain and substitution variants (Table 1 ), purified enzyme (< 5 mg/mL) was incubated at 75°C for up to 4.5 h in 20 mM tri-sodium citrate dihydrate, 150 mM NaCl, pH 5.5 (at RT) with 2.5 mM DTT and 1 mM CaCl2, respectively (activation buffer). To investigate if globupain could in-trans activate, 10 μg of activated WT globupain was mixed with 10 μg of inactive C185A variant. The number and size of cleavage products were assessed by visualization of protein bands on 8%–16% SurePAGE precast gels (GenScript) using MES SDS running buffer (GenScript) in a Bio-Rad Mini-PROTEAN Tetra Cell (BioRad, Hercules, CA, United States). For sample preparation, 4′ lithium dodecyl sulfate (LDS) sample buffer (GenScript) with 2-mercaptoethanol was mixed with the protein sample, followed by denaturing at 95°C for 10 min. Gels were stained with InstantBlue™ ultrafast protein stain (Abcam, Cambridge, United Kingdom), and the size of bands was indicated by broad multi-color pre-stained protein standard (GenScript). Edman sequencing was performed on a Shimadzu PPSQ-53A at the Iowa State University Protein Facility, United States.
3
Western Blot Analysis of Retinal Proteins
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Proteins were extracted from tissue homogenates. For Western blot analysis, protein samples were fractionated in 4–20% SurePAGE™ Precast Gels (M00657, GenScript Biotech Corporation, China) and transferred to nitrocellulose membranes. The analysis was performed with anti-CCN1 (ab228592, Abcam, UK), anti-CDC42 (2466T, Cell Signaling Technology, USA), anti-Claudin5 (4C3C2, Invitrogen, USA), anti-COX6c (ab150422, Abcam, UK), anti-CREB1 (R23983, ZEN- BIOSCIENCE, China), anti-HIF1α (ab179483, Abcam, UK), anti-NDUFα1 (15561-1-AP, Proteintech Group, USA), anti- SEPN1 (55333-1-AP, Proteintech Group, USA), anti-SHP1 (ab32559, Abcam, UK, anti-VEGFa (NB100-664, Novus Biologicals, USA), and anti-β-tubulin (2146S, Cell Signaling Technology, USA) antibodies. Immunoassays were performed using enhanced chemiluminescence (17046; ZEN-BIOSCIENCE, China), in accordance with the manufacturer’s instructions. Protein levels were determined by densitometric scanning of protein bands. Six retinas were used in each group, and the results for each retina were averaged from three replicates.
4
Comprehensive Protein Expression Analysis
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Cells were harvested and lysed with M-PER lysis buffer (Thermo Scientific) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Beyotime Biotechnology) for 30 min at 4°C. Protein concentration was determined using the BCA protein quantification kit (Beyotime Biotechnology). Equal amounts of the proteins were separated by SurePAGE™ precast gels with a linear gradient between 4%-20% (GenScript, Nanjing, China) and transferred to PVDF membranes (Millipore, Billerica, MA, USA) by eBlot® L1 protein transfer system (GenScript). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against β-actin (Beyotime Biotechnology), Pol ι (Proteintech, Rosemont, IL, USA), G6PD (Abcam, Cambridge, MA, USA), OGT (Proteintech), O-GlcNAc (Invitrogen Life Technologies, Carlsbad, CA, USA), Erk and p-Erk (Cell Signaling Technology, Danvers, Massachusetts, USA) at 4°C overnight. After washing with TBST three times, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (MultiSciences, Hangzhou, China). High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China) was applied for band visualization. Images of the protein bands were collected by Tanon-5200 Chemiluminescent Imaging System (Tanon). β-actin expression was served as a loading control.
5
Quantitative Analysis of ClpXP-Mediated Proteolysis
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ClpXP-mediated proteolysis reactions were carried out in PD buffer supplemented with 5 mM ATP, 16 mM creatine phosphate, and 0.32 mg/ml creatine kinase, 0.3 μM ClpX6, and 0.9 μM ClpP14, and incubated at 30 °C for 2 min before the addition of 3 μM of substrate protein to the reaction mixture (25 (link)). Sample aliquots (10 μl) were collected from the reaction mixture at various intervals, and the reaction was quenched by adding SDS loading dye. Samples were flash-frozen and stored at −30 °C. All the samples collected at various time points were loaded and separated on 12% SurePAGE precast gels (GenScript) and stained with the Oriole fluorescent gel stain (Bio-Rad) using the protocol provided by the manufacturer. The gels were imaged using a gel documentation system (ChemiDoc Imaging system, Bio-Rad). The amount of substrate protein band intensities was quantified using Bio-Rad ImageLab software. All the experiments were done in triplicates and presented as the mean values with standard deviation.
6
Western Blot Analysis of Brain and Liver Proteins
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TMAO and vehicle-control treated slices post-incubation control (wild-type) mice, 3×Tg-AD, and db/db mice brains were lysed in N-PER containing a protease and phosphatase inhibitor cocktail (Halt Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific, Waltham, MA, USA). The liver lysates from control (wild-type) mice, 3×Tg-AD, and db/db mice were lysed in cell lysis buffer with Halt Protease and Phosphatase Inhibitor Cocktail). Total protein was estimated by bicinchoninic acid assay-BCA assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA) and then stored at −80°C until use. Samples were mixed thoroughly with 4× Laemmli buffer (Alfa Aesar) and were loaded into 8–16% SurePAGE precast gel (GenScript Biotech). The proteins were transferred to nitrocellulose membranes (Immobilon-p Millipore, Darmstadt, Germany) and blocked with 5% Bovine serum albumin in Tris-buffered saline, 0.1% v/v Tween 20 (TBST) for 1 h. Membranes were washed with TBST and incubated with primary antibodies listed in Table 1 . Membranes were probed with secondary anti-rabbit or anti-mouse antibody (Cell Signaling Technology, Danvers, MA, USA 1:5,000) for 1 h. Membranes were imaged with Biorad imager. Finally, the densities of these bands were normalized to GAPDH/alpha-tubulin.
7
Western Blot Analysis of Pluripotent Cells
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Human fibroblasts, ESCs, prEpiSCs, and ci8CLCs cultured on 6-well plate were washed twice with ice-cold DPBS and then lysed on ice in extraction buffer containing 50 mM Tris-base (pH 7.4), 100 mM NaCl, 1% NP-40, 10 mM EDTA, 20 mM NaF, 1 mM PMSF, 3 mM Na 3 VO4, and protease inhibitors. Protein samples were separated by 4-20% SurePAGE precast gel (Genscript, cat#M00656) and then transferred to nitrocellulose membranes (Millipore, catalog. no. HATF00010). After blocking with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for one hour at room temperature, transferred membranes were incubated overnight at 4 C with different primary antibodies against b-Actin (1:2000 dilution) or LEUTX (1:1000 dilution). After horseradish peroxidase-conjugated secondary antibody incubation, bands were visualized using a ChemiDoc Touch Imaging System (Bio-Rad).
8
Verification of Nup96 Antibody Specificity
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The peptide (amino acid residues from 505 to 959) of Nup96 protein was used as the antigen to develop Nup96specific antibody by Abmart. To verify the specificity of Nup96 antibody, 12-d-old seedlings of Col-0, nup96-1, and nup96-2 were collected and ground to fine powder in liquid nitrogen. Three volumes of extraction buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, and 1× protease inhibitor mixture [Roche]) was added to the sample. After homogenization on ice for 20 min, the mixture was centrifuged at 13,000g at 4°C for 10 min. The supernatant was transferred to a new tube, 10% of which was saved as "input" sample. IP was performed as follows: 5 μL of Nup96 antibody was added to the cleared protein extracts. After incubation at 4°C for 3 h, 30 μL of Protein A/G-agarose (Abmart, A10001) was added and incubated for another 2 h at 4°C. After washing Protein A/G-agarose with extraction buffer (without protease inhibitor cocktail) five times, the proteins were eluted with 2× SDS-PAGE loading buffer by heating at 95°C for 5 min and separated on a 4 to 20% SurePAGE precast gel (Genscript, M00657) followed by immunoblot analyses.
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