Anti-ARHGEF5 was generated in rabbits via immunization with GST-mouse or human ARHGEF5 (amino acids 2–204) and affinity purified using a maltose-binding protein-tagged antigen. Anti-Src-pY418, anti-GFP, anti-FAK-pY397, SD208, Alexa Fluor 488 phalloidin, Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G, horse radish peroxidase-conjugated goat anti-rabbit immunoglobulin G, anti-mouse immunoglobulin G, anti-occludin and anti-cortactin-pY421 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-GAPDH, anti-Fyn, anti-Lyn and anti-vimentin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-v-Src, anti-cortactin (4F11) and anti-phosphotyrosine (4G10) were from Millipore (Billerica, MA, USA). Anti-FLAG (M2) and anti-β-tubulin were from Sigma-Aldrich (St Louis, MO, USA). Anti-E-cadherin, anti-N-cadherin and anti-FAK were from BD Transduction Laboratories (Lexington, KY, USA). anti-Smad2-pS465/467, anti-Smad2, anti-MLC2-pT18/S19, anti-MLC2, anti-Akt and anti-Akt-pS473 were from Cell Signaling Technology Inc. (Beverly, MA, USA). TGF-β1 and TNF-α were from PeproTech (Rocky Hill, NJ, USA). The Akt inhibitor triciribine was from Selleckchem (Houston, TX, USA).
Horseradish peroxidase conjugated goat anti rabbit immunoglobulin g
Manufactured by Thermo Fisher Scientific
Sourced in United States
Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G is a secondary antibody used in various immunoassay techniques. It consists of goat-derived antibodies that specifically bind to rabbit immunoglobulin G, with horseradish peroxidase enzyme molecules attached to them.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti py397 fak, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Anti ps473 akt, by Cell Signaling Technology (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti n cadherin, by BD (1 mentions)
Anti fak, by BD (1 mentions)
Anti cortactin 4f11, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti phosphotyrosine 4g10, by Merck Group (1 mentions)
Triciribine, by Selleck Chemicals (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti lyn, by Santa Cruz Biotechnology (1 mentions)
Anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Anti mlc2, by Cell Signaling Technology (1 mentions)
3 protocols using horseradish peroxidase conjugated goat anti rabbit immunoglobulin g
1
Immunoprecipitation and Western Blot Analysis
2
Western Blot Analysis of NRF2 Protein
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Protease inhibitors (Boster, Wuhan, China) were added to cell lysates, which were maintained on ice for 20 minutes. Lysates were then centrifuged at 12,000 rpm for 10 min at 4°C. Samples (50 μg) were boiled for 5 min in sample buffer and then separated on 12% gels by SDS-PAGE. Gels were transferred onto nitrocellulose membranes and blocked for 1 h in 5% skim milk at room temperature with shaking. A primary antibody against NRF2 (Abcam, USA) or β-Actin (Sangon, Shanghai, China) was added overnight to blots at 4°C. Blots were washed in PBS-Tween three times, after which the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G; Thermo, IL, USA) was added at room temperature for 2 hours. Chemiluminescent substrate (Thermo, IL, USA) was added to visualize bands. Quantity One software was used to quantify the intensity of each band and was normalized to the intensity of the internal control β-Actin. Results were expressed as fold changes normalized to control values.
3
Western Blot Quantification of Signaling Proteins
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Western blot was performed as previously described (19 (link),20 (link)). Briefly, 40 µg extracted protein samples were transferred onto a polyvinylidene difluoride (EMD Millipore) membrane and then blocked with 5% skim milk for 2 h at 4°C. Then each membrane was probed with primary antibodies against PTEN, phosphorylated-protein kinase B (p-Akt) (S473) (cat. no. OMA1-03061; 1:1,000), Akt (cat. no. 44-609G; 1:1,000), p-glycogen synthase kinase (GSK)-3β (S9) (cat. no. MA5-14873; 1:1,000), GSK-3β (cat. no. MA3-038; 1:1,000), β-catenin (cat. no. 71-2700; 1:1,000), c-Myc (cat. no. MA5-27025; 1:1,000), matrix metallopro-teinase-9 (MMP-9; cat. no. MA5-15886; 1:1,000) and β-actin (cat. no. MA5-15739-D800; 1:1,000) (all primary antibodies were obtained from Thermo Fisher Scientific, Inc.) at 4°C for 20 h, followed by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:10,000; cat. no. 205718; Abcam). β-actin served as the loading control and for normalization of protein expression. The protein bands were developed using ECL kit (GE Healthcare) and blot bands were quantified with ImageJ (version 1.46; Rawak Software, Inc.).
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