COS-7 cells and HEK 293T cells were cultured and transiently transfected as indicated with mouse NTPDase8 or human NTPDase8 cDNA constructs as described previously (Kukulski et al., 2005 (link)). For Western blot assays, lysates from transfected or non-transfected COS-7 cells (6 μg, unless otherwise indicated) were resuspended in NuPAGE sample buffer, separated on NuPAGE 4–12% Bis-Tris gels under reduced or non-reduced conditions, as indicated, and transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA) by electroblotting according to the manufacturer’s recommendation. Membranes were then blocked with 2.5% non-fat milk in PBS containing 0.15% Tween20®(pH 7.4) O/N at 4°C and subsequently probed with the primary antibodies. Appropriate secondary HRP-conjugated antibodies were used, and the membranes developed with the Western LightningTM Plus-ECL system (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). In some experiments, a gel with a large sample well 6 cm long containing 120 μg of lysates, transferred and blocked as described above and then probed with antibodies using the Mini-Protean II multiscreen apparatus (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada) in which 20 antibodies can be tested on one gel.
Mini protean 2 multiscreen apparatus
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN II Multiscreen Apparatus is a laboratory equipment used for performing gel electrophoresis. It allows for the simultaneous separation and analysis of multiple protein or nucleic acid samples. The device features a compact design and provides a convenient platform for conducting multiple gel runs in parallel.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Vwr200 homogenizer, by Avantor (2 mentions)
Peroxidase conjugated affinipure goat anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
X ray film, by Cytiva (1 mentions)
Western lightning plus ecl detection reagent, by PerkinElmer (1 mentions)
Goat anti mouse igg hrp, by Southern Biotech (1 mentions)
Goat anti mouse iga hrp, by Southern Biotech (1 mentions)
Anti zo 1, by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Western ecl substrate, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti occludin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
25 protocols using mini protean 2 multiscreen apparatus
1
Western Blot Analysis of NTPDase8 in Transfected Cells
2
Serum Antibody Reactivity Analysis
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Serum IgG or IgA reactivity toward CBL or HhL was analyzed by immunoblot analysis. For screening multiple sera from Ctr and DC-LMP1/CD40 animals, the Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad, Cat: 1704017) was used. CBL (30 µg per lane or 600 µg for Mini-PROTEAN II Multiscreen Apparatus) or HhL (20 µg per lane or 200 µg for Mini-PROTEAN II Multiscreen Apparatus) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Sera of mice were used as primary antibodies. Differences in serum antibody concentrations between Ctr and DC-LMP1/CD40 mice were adjusted by using 2.5 µg ml−1 serum IgG or 1 µg ml−1 serum IgA for all samples. In some experiments, mouse IgG1 anti-human heat shock protein 60 (aHSP60) antibody (clone LK-2, Enzo, Cat: ADI-SPA-807-E) was additionally used as the primary antibody (1:10,000 in PBS/1% nonfat dried milk). HRP-conjugated secondary antibodies were used as follows: goat anti-mouse IgG-HRP (SouthernBiotech, Cat: 1030–05; 1:10,000) or goat anti-mouse IgA-HRP (SouthernBiotech, Cat: 1040–05; 1:10,000). Western Lightning Plus-ECL Detection Reagent (PerkinElmer, Cat: NEL103E001EA) and X-ray films (Amersham, Cat: 45–001-504) were used for protein detection.
3
Screening Patient Sera for PLA2R-Ab
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Patient sera were screened for PLA2R-Ab as previously reported using the Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad) (13 (link)). Briefly, nitrocellulose membranes blotted with 1D4-tagged PLA2R full-length or PLA2R CysR-CTLD1 triple domain (dominant-epitope) were assembled into the multiscreen apparatus after 30 min of blocking with TBSTM buffer. Each patient serum was applied at 1:100 dilutions (in TBSTM) on the membranes and incubated for 2 h at room temperature. The membranes were then washed three times with TBST buffer and incubated with HRP-conjugated goat anti-human IgG antibodies in TBSTM buffer at a dilution of 1:5000. The 1D4-Ab was included as a positive control, detected by a secondary HRP-conjugated goat anti-mouse IgG. The membranes were then incubated with ECL reagent for 1 min and exposed to Amersham Highperfilm ECL. The exposure times were 1 min for positive bands and up to 5 min for weak or negative bands. In separate experiments, patient sera were applied at 1:100, 1:1000, or 1:5000 dilutions onto the membranes, and the films were exposed for 5 to 8 min for the positive bands.
4
Western Blot Analysis of Tight Junction Proteins
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Total protein was extracted from Caco-2 cells using lysis buffer, and protein concentrations were adjusted to a known concentration using Bradford reagent (Sigma, St. Louis, MO, USA), before conducting electrophoresis. TJ proteins were resolved on 8% (for occludin and ZO-1) and 15% (for Claudin-4) SDS–polyacrylamide mini-gels using a Mini-PROTEAN® II Multiscreen Apparatus (Bio-Rad Laboratories, Hercules, CA, USA), and then transferred to nitrocellulose membranes (0.2 μm; Bio-Rad). Non-specific binding was ensured using 5% BSA, and membranes were incubated with anti-human primary antibodies, including anti-Claudin 4, anti-occludin, anti-ZO-1, and anti-GAPDH (Sigma, St. Louis, MO, USA) at 4 °C overnight in the dark. Membranes were washed three times with Tris-buffered Saline-Tween (TBST) and incubated with secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA), for 60 min. The membranes were washed three times with TBST, reacted against a Western ECL substrate (Bio-Rad Laboratories (Canada) Ltd., Mississauga, ON, Canada) for 5 min, and analyzed using the ChemiDocTM MP Imaging System (Bio-Rad Laboratories Ltd., Hercules, CA, USA).
5
Western Blot Analysis of Autoantibodies in dKO Mice
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Pancreas, salivary glands and kidney from female Rag1−/−mice were homogenized (VWR200 Homogenizer, VWR) on ice in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100,1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and a protease inhibitor cocktail (Thermo Scientific). The homogenates were centrifuged at 12,000 x g per minute for 10 min, and the supernatants were mixed with a 4 x Laemmli Sample Buffer (Bio-Rad) and boiled for 5 min at 100°C. The extracts were separated by 10% SDS-PAGE gel and electrophoretically transferred onto a PVDF membrane (Bio-Rad). The membranes were blocked with PBS (VWR) containing 0.1% Tween-20 (Fisher Scientific) and 5% nonfat milk (Bio-Rad) for 1 h and assembled in a Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad) and incubated overnight with 1:600 dilution of sera collected from dKO or control mice. After washing three times with PBST, the bound antibodies were reacted with Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (1:5,000, Jackson ImmunoResearch Laboratories) for 1 h, washed with PBST for three times, revealed with Clarity Western ECL Substrate (Bio-Rad) and autoradiography.
6
Western Blot Analysis of Autoantibodies in dKO Mice
7
Recombinant Protein Immunoblotting Screening
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The purified recombinant His-tag KP1_p307 protein was separated on 12% SDS-PAGE (~5 ng per lane) and then transferred onto a membrane. Patients’ sera (1:2000 dilution) were screened simultaneously using Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad, Hercules, CA). Those collected first were screened first, and immunoblotting with serial sera was performed when the first collected serum was antibody negative.
8
Immunoblotting of Aire-Deficient Serum
9
Western Blot Analysis of Salivary Proteins
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Nymphal (5th instar) or adult salivary proteins (50 µg protein in a 2D well) were separated by SDS-PAGE as described above and transferred onto a nitrocellulose membrane at 25 V for 30 min using the Pierce Fast Semi-dry Blotter (Thermo Scientific). After the transfer, the membrane was blocked with 5% skimmed milk in PBS overnight at 4°C, washed three times in PBST and incubated with guinea pig sera diluted 1∶100 in PBST with 5% skimmed milk for 1 h at room temperature using the Mini-Protean II Multiscreen apparatus (Bio-Rad). Following repeated washing steps, the Western blot membrane was incubated for 1 h at room temperature with HRP conjugated rabbit anti-guinea pig IgG secondary antibodies (Sigma-Aldrich) diluted 1∶10,000 in PBST with 5% dried skimmed milk or HRP goat anti-guinea pig IgM secondary antibodies (Immunology Consultants Laboratory), diluted 1∶5,000 in PBST with 5% dried skimmed milk. The proteins were visualized using the Pierce ECL Western Blotting Substrate (Thermo Scientific) and digitized using the LAS-3000 machine (Fujifilm).
10
Enzyme Encapsulation Efficiency Determination
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ASNase-loaded NPs were submitted to three cycles of centrifugation at 3220 g for 15 min and resuspended in 5.0 ml of PBS, pH 7.4, to remove any non-encapsulated molecule of the enzyme. The pellet was dissolved in 1.0 ml of 50% dimethyl sulfoxide (DMSO) in PBS (v/v). and the enzyme concentration quantified as total proteins using a BCA Kit. ASNase encapsulation efficiency (EE % ) was calculated according to the equation:
where m p0 is the initial mass of protein and m p the mass of protein in the pellet solution. The pellet solution was also subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a Mini-PROTEAN II Multiscreen apparatus (Bio-Rad Laboratories, Hercules, CA, USA) and the gel stained with Coomassie blue. The protein molecular weight marker was acquired from Bio-Rad Laboratories (Hercules, CA, USA). Another electrophoretic separation with retention of native properties (Native-SDS-PAGE) was performed to evaluate the enzymatic activity in this pellet solution through zymogram according to the L-aspartyl-β-hydroxamic acid method. According to this method, L-aspartyl-β-hydroxamic acid is hydrolyzed by ASNase, producing aspartic acid, hydroxylamine and red hydroxamic acids complexes with ferric chloride absorbing at 490 nm.
where m p0 is the initial mass of protein and m p the mass of protein in the pellet solution. The pellet solution was also subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a Mini-PROTEAN II Multiscreen apparatus (Bio-Rad Laboratories, Hercules, CA, USA) and the gel stained with Coomassie blue. The protein molecular weight marker was acquired from Bio-Rad Laboratories (Hercules, CA, USA). Another electrophoretic separation with retention of native properties (Native-SDS-PAGE) was performed to evaluate the enzymatic activity in this pellet solution through zymogram according to the L-aspartyl-β-hydroxamic acid method. According to this method, L-aspartyl-β-hydroxamic acid is hydrolyzed by ASNase, producing aspartic acid, hydroxylamine and red hydroxamic acids complexes with ferric chloride absorbing at 490 nm.
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