For western blot analysis, the cells were lysed in radioimmunoprecipitation assay buffer (RIPA) buffer (50 × 10−3m Tris‐HCl, 150 × 10−3m NaCl, 5 × 10−3m ethylenediaminetetraacetic acid (EDTA), 0.5% NP‐40) containing protease inhibitor and phosphatase inhibitor cocktails (Thermo scientific). Lysates were cleared by centrifugation at 12 000 rpm for 20 min at 4 °C. The lysates were first incubated with antibody beads or agarose overnight at 4 °C, or antibodies overnight at 4 °C followed by incubation with protein A/G PLUS‐Agarose (Santa Cruz) at 4 °C for 2 h. Then the precipitates were washed five times with cold RIPA buffer and eluted with 5 x SDS sample buffer. The immunoprecipitates were separated by SDS‐PAGE and transferred to a polyvinylidene fluoride membrane (Millipore). The membranes were blocked in Tris‐buffered saline (TBS) with 5% nonfat milk and 0.1% Tween 20, probed with primary antibodies overnight at 4 °C, washed five times by TBS with 5% nonfat milk and 0.1% Tween 20, and then incubated with secondary horseradish‐peroxidase‐conjugated antibodies (Promega). ClarityTM Western ECL substrate (Bio‐Rad) was then used for detection.
Secondary horseradish peroxidase conjugated antibodies
Manufactured by Promega
Sourced in United States
Secondary horseradish peroxidase conjugated antibodies are laboratory reagents used to detect and quantify primary antibodies in various immunoassays. The horseradish peroxidase enzyme allows for colorimetric or chemiluminescent detection of the bound antibody complex.
Automatically generated - may contain errors
Lab products found in correlation
Anti atsec10, by Promega (2 mentions)
Anti atsec15a, by Promega (2 mentions)
Rabbit polyclonal anti gfp, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Irs 1, by Merck Group (1 mentions)
Phospho smad2, by Cell Signaling Technology (1 mentions)
Phospho akt thr308, by Cell Signaling Technology (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
4 protocols using secondary horseradish peroxidase conjugated antibodies
1
Immunoprecipitation and Western Blot Analysis
2
Western Blot Analysis of Adipocyte Proteins
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Cells were rinsed with PBS and solubilized in stop buffer containing 50 mM Hepes, pH 7.2, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 2 mM Na3VO4, and 1% Triton X-100 supplemented with Protease Inhibitor Cocktail (Roche). The primary antibodies and dilutions used were: UCP1 (1/500, Calbiochem), Perilipin 1 (1/1000, Acris Antibodies); Tubulin (1/10000, Sigma); AKT (1/1000, Cell Signaling); Phospho-AKT (Thr308) (1/1000, Cell Signaling); Phospho-Erk1/2 (Thr202/Tyr204) (1/1000, Cell Signaling); Phosphotyrosin, clone 4G10 (1/1000, Millipore); IRS1 (1/1000, Millipore); Phospho-Smad2 (1/1000, Cell Signaling); Smad 2/3 ((1/1000, Cell Signaling). Secondary horseradish peroxidase-conjugated antibodies were purchased from Promega.
3
Exocyst complex protein analysis
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Total protein extracts were isolated from 2-4-week-old plants transformed with EXO70B2-GFP at different time points ranging from 0-24 hpi with Bg or water (mock). The protein extraction Sec6/8 buffer adjusted for exocyst extraction was used (20 mM HEPES, pH 6.8, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.5% Tween, supplemented with protease inhibitor cocktail (Sigma-Aldrich)). After one hour of lysis, the extracts were spun down, and the supernatants were boiled in 6x SDS loading buffer. The proteins were loaded for 10% SDS-PAGE and blotted onto a nitrocellulose membrane, with 20 ng per lane. The membrane was stained with Ponceau S solution and blocked overnight with 5% nonfat dry milk in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4, 0.25% Tween 20). Primary antibody dilutions in PBS were as follows: polyclonal rabbit anti-GFP (Invitrogen), 1:800; polyclonal rabbit antibodies anti-AtSEC3, 1:5000; anti-AtSEC5, 1:5000; anti-AtSEC6 (Agrisera Sweden), 1:10000; anti-AtSEC8 (Agrisera Sweden), 1:8000; anti-AtSEC10, 1:10000; anti-AtSEC15a, 1:1000 and anti-AtEXO84b, 1:1000.
Appropriate secondary horseradish peroxidase conjugated antibodies (Promega, Madison, WI, USA) were applied, followed by chemiluminescent ECL detection (Amersham, GE Healthcare, Chicago, IL, USA).
Appropriate secondary horseradish peroxidase conjugated antibodies (Promega, Madison, WI, USA) were applied, followed by chemiluminescent ECL detection (Amersham, GE Healthcare, Chicago, IL, USA).
4
Exocyst complex protein analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extracts were isolated from 2-4-week-old plants transformed with EXO70B2-GFP at different time points ranging from 0-24 hpi with Bg or water (mock). The protein extraction Sec6/8 buffer adjusted for exocyst extraction was used (20 mM HEPES, pH 6.8, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.5% Tween, supplemented with protease inhibitor cocktail (Sigma-Aldrich)). After one hour of lysis, the extracts were spun down, and the supernatants were boiled in 6x SDS loading buffer. The proteins were loaded for 10% SDS-PAGE and blotted onto a nitrocellulose membrane, with 20 ng per lane. The membrane was stained with Ponceau S solution and blocked overnight with 5% nonfat dry milk in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4, 0.25% Tween 20). Primary antibody dilutions in PBS were as follows: polyclonal rabbit anti-GFP (Invitrogen), 1:800; polyclonal rabbit antibodies anti-AtSEC3, 1:5000; anti-AtSEC5, 1:5000; anti-AtSEC6 (Agrisera Sweden), 1:10000; anti-AtSEC8 (Agrisera Sweden), 1:8000; anti-AtSEC10, 1:10000; anti-AtSEC15a, 1:1000 and anti-AtEXO84b, 1:1000.
Appropriate secondary horseradish peroxidase conjugated antibodies (Promega, Madison, WI, USA) were applied, followed by chemiluminescent ECL detection (Amersham, GE Healthcare, Chicago, IL, USA).
Appropriate secondary horseradish peroxidase conjugated antibodies (Promega, Madison, WI, USA) were applied, followed by chemiluminescent ECL detection (Amersham, GE Healthcare, Chicago, IL, USA).
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