SDS–PAGE was performed on Bio-Rad Mini-PROTEAN systems using standard protocols. For immunostaining, proteins were transferred onto 0.2-µm nitrocellulose membranes (Amersham Protran). Immunoblots were probed with primary antibodies and goat secondary antibodies coupled to alkaline phosphatase, and developed in alkaline buffer in the presence of 5-bromo-4-chloro-3-indolylphosphate and nitro-blue tetrazolium. The anti-HA (HA-7 clone, Sigma Aldrich), anti-FLAG (M2 clone, Sigma Aldrich), anti-StrepII (Sigma Aldrich), anti VSV-G (Sigma Aldrich) and anti-5His (Sigma Aldrich) monoclonal antibodies, and mouse secondary antibodies (Millipore) were purchased as indicated.
Mouse secondary antibodies
Manufactured by Merck Group
Mouse secondary antibodies are laboratory reagents used in immunoassay techniques. They are designed to bind to primary antibodies raised in mice, allowing for the detection and visualization of target antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag clone m2, by Merck Group (3 mentions)
Anti strepii, by Merck Group (3 mentions)
Anti 5his, by Merck Group (3 mentions)
Anti ha clone ha 7, by Merck Group (2 mentions)
Anti vsv g, by Merck Group (2 mentions)
Protran, by Cytiva (2 mentions)
Mini protean, by Bio-Rad (2 mentions)
G box chemi xt4 device, by Syngene (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Tween 20, by Merck Group (2 mentions)
Mini protean system, by Bio-Rad (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Mouse anti β actin primary antibody, by Merck Group (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using mouse secondary antibodies
1
Immunoblotting Using Common Antibodies
2
Western Blot Analysis Protocol
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SDS-PAGE was performed on Bio-Rad Mini-Protean systems using standard protocols with homemade 12.5% polyacrylamide gels. For immunostaining, proteins were transferred onto 0.2-μm nitrocellulose membranes (Amersham Protran) with a Mini-Trans Blot cell (Bio-Rad). The membrane was then saturated in 5% milk and probed with primary antibodies. Mouse secondary antibody coupled to alkaline phosphatase was added and developed in alkaline buffer in the presence of 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium. The antihemagglutinin (HA) (HA-7 clone; Sigma-Aldrich), anti-Flag (M2 clone; Sigma-Aldrich), anti-StrepII (Sigma-Aldrich), anti-5His (Sigma-Aldrich) monoclonal antibodies, and mouse secondary antibodies (Millipore) were purchased as indicated.
3
Immunoblotting Techniques for Protein Detection
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SDS-PAGE was performed on Bio-Rad Mini-PROTEAN ® systems using standard protocols. For immunostaining, proteins were transferred onto 0.2-µm nitrocellulose membranes (Amersham Protran). Immunoblots were probed with primary antibodies and goat secondary antibodies coupled to alkaline phosphatase, and developed in alkaline buffer in presence of 5-bromo-4-chloro-3indolylphosphate and nitro-blue tetrazolium. The anti-HA (HA-7 clone, Sigma Aldrich), anti-Flag (M2 clone, Sigma Aldrich), anti-StrepII (Sigma Aldrich), anti VSV-G (Sigma Aldrich) and anti-5His (Sigma Aldrich) monoclonal antibodies, and mouse secondary antibodies (Millipore) were purchased as indicated.
4
Western Blot Analysis of hOE-MSC Proteins
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For analyses by western blotting, hOE-MSCs were collected by trypsination, counted and then centrifuged for 5 min at 300 g . Cell pellets were resuspended in 1× SDS loading buffer, heated at 95°C during 5 min, then each cell lysate was resolved by 8% SDS-PAGE and transferred onto a PVDF membrane (Thermo Scientific, Rockford, IL). After blocking membrane with 5% milk in PBS (Life Technologies, Gibco, Grand Island, NY) supplemented with 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MO) (PBS-T), membrane was incubated at 4°C overnight with mouse monoclonal primary antibodies against IKAP diluted at 1:2000 (clone no 33, BD Biosciences, Franklin Lakes, NJ), anti-NOVA1 at 1:300 (cat. no WH0004857M7, Sigma-Aldrich, St. Louis, MO) or anti-β-actin at 1:10,000 (cat. no A2228, Sigma-Aldrich, St. Louis, MO) in 2.5% milk in PBS-T. Thereafter, membrane was then probed with mouse secondary antibodies diluted at 1:3500 (Sigma-Aldrich, St. Louis, MO) in 2.5% milk in PBS-T at room temperature for 45 min, and revealed by exposing pre-incubated membrane with ECL (ThermoScientific, Rockford, IL) using a G:BOX Chemi XT4 device (Syngene, Cambridge, UK). β-actin was used for normalization as the housekeeping protein.
5
Western Blot Analysis of IKAP/hELP1
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Cell pellets were resuspended in 1X SDS loading buffer at 6x10 3 cells/µL, and then samples were heated at 95°C during 5 min. Thereafter, total protein from 10 µl of cell lysates were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Thermo Fisher Scientific, Rockford, IL). Membranes were blocked with 5% Milk in PBS supplemented with 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MO) buffer for 1h at room temperature, and were then subsequently probed at 4°C overnight with mouse monoclonal anti-IKAP/hELP1 antibody diluted at 1:2000 (BD Biosciences, Franklin Lakes, NJ) in 2.5% Milk in PBST, followed by incubation with mouse secondary antibodies at 1:3500 (Sigma-Aldrich). As a control of equal protein loading, membranes were also probed with mouse anti-β-actin primary antibody used at 1:10000 (Sigma-Aldrich). Stained proteins were revealed by chemiluminescent detection using ECL detection kit (Thermo Fisher Scientific, Rockford, IL) on a G:BOX Chemi XT4 device (Syngene, Cambridge, UK).
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