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Trueblot secondary antibody

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TrueBlot secondary antibodies are a line of highly specific and sensitive detection reagents designed for immunoassays. They are engineered to minimize background signal and provide reliable results in Western blotting and other immunodetection applications.

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Lab products found in correlation

Horseradish peroxidase conjugate secondary antibody, by Bio-Rad (2 mentions) Acrylamide gel, by Bio-Rad (2 mentions) Ecl plu, by Cytiva (2 mentions) Lowry protein assay, by Bio-Rad (2 mentions) Protran, by Cytiva (2 mentions) Hrp conjugated goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions) Goat anti mouse igg, by Jackson ImmunoResearch (2 mentions) Trip13, by Proteintech (2 mentions) Anti phospho h2ax ser139 γh2ax, by Merck Group (2 mentions) Anti phospho dna pkcs ser 2056, by Cell Signaling Technology (2 mentions) Dna pkcs, by Cell Signaling Technology (2 mentions) Anti gfp antibody, by Santa Cruz Biotechnology (1 mentions) Phospho s6, by Cell Signaling Technology (1 mentions) Anti v5 antibody, by Thermo Fisher Scientific (1 mentions) Protein a sepharose beads, by Cytiva (1 mentions) Advanced ecl detection system, by Cytiva (1 mentions)

6 protocols using trueblot secondary antibody

1

Antibody Procurement for GFP, HA, and Phospho-Proteins

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Anti -GFP antibodies (# 9996) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The HA antibody was obtained from Sigma (Newark, NJ). S6, Phospho-S6, YAP and Phospho-YAP antibodies (#2212, #2211, #4912, #4911) were obtained from Cell Signaling (Danvers, MA) Trueblot secondary antibodies were purchased from eBioscience (San Diego, CA).HRP conjugated or Trueblot secondary antibodies were purchased from eBioscience (San Diego, CA).
Nelson N, & Clark G.J. (2016). Rheb may complex with RASSF1A to coordinate Hippo and TOR signaling. Oncotarget, 7(23), 33821-33831.
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2

Protein Extraction and Western Blot Analysis

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Cell lysates were prepared with RIPA Buffer. Mouse tissues were homogenized mechanically in tissue lysis buffer (50mM Tris HCl pH 7.5, 320mM Sucrose, 50mM NaCl, 1% Triton X-100 and protease and phosphatase inhibitors). Homogenates were centrifuged at 13,000xrpm at 4°C for 30min and supernatants were collected. Protein extracts were quantified by Lowry protein assay (Biorad), and denatured by adding Laemmli Buffer (0,1% Bromophenol blue, 10% Glycerol, 2% SDS, 5% ß-mercaptoethanol, 270mM Tris HCl pH.6.8). A total of 30-50μg of proteins was analysed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Proteins were separated on acrylamide gel (Biorad) and electroblotted onto nitrocellulose membranes (Protran, Schleicher & Schuell). Blots were incubated with primary antibodies in 5% non-fat dry milk in PBS plus 0,1% Tween-20 overnight at 4°C. Detection was achieved using horseradish peroxidase-conjugate secondary antibody (Biorad) and visualized with ECL plus (Amersham Bioscience). For the Western Blot analysis of immunoprecipitated C-MYC, TrueBlot@ secondary antibody (EBioscience) was used, in order to minimize detection of Immunoglobulines in the immunoprecipitation samples.
Cianfanelli V., Fuoco C., Lorente M., Salazar M., Quondamatteo F., Gherardini P.F., De Zio D., Nazio F., Antonioli M., D’Orazio M., Skobo T., Bordi M., Rohde M., Dalla Valle L., Helmer-Citterich M., Gretzmeier C., Dengjel J., Fimia G.M., Piacentini M., Di Bartolomeo S., Velasco G, & Cecconi F. (2014). AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-MYC dephosphorylation and degradation. Nature cell biology, 17(1), 20-30.
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3

Protein Extraction and Western Blot Analysis

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Cell lysates were prepared with RIPA Buffer. Mouse tissues were homogenized mechanically in tissue lysis buffer (50mM Tris HCl pH 7.5, 320mM Sucrose, 50mM NaCl, 1% Triton X-100 and protease and phosphatase inhibitors). Homogenates were centrifuged at 13,000xrpm at 4°C for 30min and supernatants were collected. Protein extracts were quantified by Lowry protein assay (Biorad), and denatured by adding Laemmli Buffer (0,1% Bromophenol blue, 10% Glycerol, 2% SDS, 5% ß-mercaptoethanol, 270mM Tris HCl pH.6.8). A total of 30-50μg of proteins was analysed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Proteins were separated on acrylamide gel (Biorad) and electroblotted onto nitrocellulose membranes (Protran, Schleicher & Schuell). Blots were incubated with primary antibodies in 5% non-fat dry milk in PBS plus 0,1% Tween-20 overnight at 4°C. Detection was achieved using horseradish peroxidase-conjugate secondary antibody (Biorad) and visualized with ECL plus (Amersham Bioscience). For the Western Blot analysis of immunoprecipitated C-MYC, TrueBlot@ secondary antibody (EBioscience) was used, in order to minimize detection of Immunoglobulines in the immunoprecipitation samples.
Cianfanelli V., Fuoco C., Lorente M., Salazar M., Quondamatteo F., Gherardini P.F., De Zio D., Nazio F., Antonioli M., D’Orazio M., Skobo T., Bordi M., Rohde M., Dalla Valle L., Helmer-Citterich M., Gretzmeier C., Dengjel J., Fimia G.M., Piacentini M., Di Bartolomeo S., Velasco G, & Cecconi F. (2014). AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-MYC dephosphorylation and degradation. Nature cell biology, 17(1), 20-30.
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4

Immunoprecipitation of SmAkt Proteins

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Expression and phosphorylation of SmAkt proteins in oocytes were confirmed by immunoprecipitation of oocyte lysates according to the procedure described previously (Vicogne et al., 2004 (link)). Following 15 h of expression, oocytes were lysed in buffer A (50 mM Hepes pH 7.4, 500 mM NaCl, 0.05% SDS, 5 mM MgCl2, 1 mg/mL bovine serum albumin, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 10 μg/mL soybean trypsin inhibitor, 10 μg/mL benzamidine, 1 mM PMSF, 1 mM sodium vanadate) containing 0.5% Triton X-100 and centrifuged at 12,000g for 15 min at 4 °C. Lysates were incubated with anti-V5 antibodies (1:100, Invitrogen) for 4 h at 4 °C. Protein A-Sepharose beads (5 mg; Amersham Biosciences) were added for 1 h at 4 °C. Beads were washed three times and resuspended in Laemmli sample buffer. Eluted immune complexes were subjected to a 10% SDS–PAGE, then analyzed by Western blotting using anti-V5 (1:50,000) or anti-phospho-T308 Akt (1:5000; Upstate Biotechnology) antibodies. Mouse or rabbit Trueblot® secondary antibodies (eBioscience) were used as secondary antibodies and chemoluminescence was revealed using the advanced ECL detection system (Amersham Biosciences).
Morel M., Vanderstraete M., Cailliau K., Lescuyer A., Lancelot J, & Dissous C. (2014). Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis. International Journal for Parasitology: Drugs and Drug Resistance, 4(3), 256-266.
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5

Immunoblotting Protein Detection Protocol

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Immunoblots were performed as described 57 (link), 58 (link). The primary antibodies were TRIP13 (1:3000, #19802-1-AP) from Proteintech, Chicago, IL, KU70 (1:1000, # AB1358), anti-phospho-H2AX(ser139) (γH2AX) (1:1000, #05–636) both from Millipore, Billerica, MA), KU80 (1:1000,#2180), DNA-PKcs (1:1000, #4602) anti-phospho-DNA-PKcs (Ser 2056) all from Cell Signaling Technology, Boston, MA, and Actin (1:3000, # 612656) from BD Biosciences, Franklin Lakes, NJ). Secondary antibodies were HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Jackson Immuno Research Laboratories, West Grove, PA). For IP, TrueBlot secondary antibodies (eBioscience Rbt#18-8816, Mouse#18-8817) were used. Immunoreactive proteins were visualized by SuperSignal-West-Pico-Chemiluminescent system (Pierce, Pittsburgh, PA). The uncropped scanned films with molecular weight reference are included in the Supplementary Figure 9 through 11 in sequence of appearance in the main text. Signal intensity was quantified using ImageJ software (http://rsbweb.nih.gov/ij/).
Banerjee R., Russo N., Liu M., Basrur V., Bellile E., Palanisamy N., Scanlon C.S., van Tubergen E., Inglehart R.C., Metwally T., Mani R.S., Yocum A., Nyati M.K., Castilho R.M., Varambally S., Chinnaiyan A.M, & D’Silva N.J. (2014). TRIP13 promotes error-prone nonhomologous end joining and induces chemoresistance in head and neck cancer. Nature communications, 5, 4527.
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6

Immunoblotting Protein Detection Protocol

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Immunoblots were performed as described 57 (link), 58 (link). The primary antibodies were TRIP13 (1:3000, #19802-1-AP) from Proteintech, Chicago, IL, KU70 (1:1000, # AB1358), anti-phospho-H2AX(ser139) (γH2AX) (1:1000, #05–636) both from Millipore, Billerica, MA), KU80 (1:1000,#2180), DNA-PKcs (1:1000, #4602) anti-phospho-DNA-PKcs (Ser 2056) all from Cell Signaling Technology, Boston, MA, and Actin (1:3000, # 612656) from BD Biosciences, Franklin Lakes, NJ). Secondary antibodies were HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Jackson Immuno Research Laboratories, West Grove, PA). For IP, TrueBlot secondary antibodies (eBioscience Rbt#18-8816, Mouse#18-8817) were used. Immunoreactive proteins were visualized by SuperSignal-West-Pico-Chemiluminescent system (Pierce, Pittsburgh, PA). The uncropped scanned films with molecular weight reference are included in the Supplementary Figure 9 through 11 in sequence of appearance in the main text. Signal intensity was quantified using ImageJ software (http://rsbweb.nih.gov/ij/).
Banerjee R., Russo N., Liu M., Basrur V., Bellile E., Palanisamy N., Scanlon C.S., van Tubergen E., Inglehart R.C., Metwally T., Mani R.S., Yocum A., Nyati M.K., Castilho R.M., Varambally S., Chinnaiyan A.M, & D’Silva N.J. (2014). TRIP13 promotes error-prone nonhomologous end joining and induces chemoresistance in head and neck cancer. Nature communications, 5, 4527.
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