Rabbit monoclonal anti akt

Manufactured by Cell Signaling Technology

Sourced in United States

The Rabbit monoclonal anti-Akt is a laboratory reagent used for the detection and analysis of the Akt protein, a key regulator of cellular processes. This antibody is produced in rabbits and specifically binds to the Akt protein, allowing researchers to study its expression and activity in various experimental systems.

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18 protocols using rabbit monoclonal anti akt

Antibody-Based Western Blotting Methodology

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Western blotting was performed as previously reported [61 (link), 62 (link)]. The antibodies used were rabbit polyclonal anti-phospho-Ser473-Akt (#9271, 1:1000, Cell Signaling Technology, Danvers MA, USA), rabbit monoclonal anti-Akt (#4691, 1:1000, Cell Signaling Technology), rabbit polyclonal phospho-Ser9-GSK3β (#9336, 1:1000, Cell Signaling Technology), rabbit monoclonal GSK3β (#9315, 1:1000, Cell Signaling Technology), rabbit polyclonal anti-phospho-Thr202/Tyr204-ERK1/2 (#9101, 1:1000, Cell Signaling Technology), rabbit polyclonal anti-ERK1/2 (#9102, 1:1000, Cell Signaling Technology), rabbit polyclonal anti-phospho-Ser217/221-MEK1/2 (#9121, Cell Signaling Technology), rabbit polyclonal anti-MEK1/2 (#9122, Cell Signaling Technology), rabbit polyclonal anti-p16 (10883-1-AP, 1:500, Proteintech, Rosemont, IL, USA), rat monoclonal anti-p19 (sc-32748, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-p21 (ab7960, Abcam, Cambridge, UK), goat polyclonal anti-IGFBP5 (sc-6006, 1:200, Santa Cruz Biotechnology), mouse monoclonal anti-FLAG (F3165, 1:1000, Sigma Aldrich; Merck KGaA, Darmstadt, Germany), mouse monoclonal anti-β-actin (010-27841, 1:10000, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and mouse monoclonal anti-GAPDH (G8795, 1:10000, Sigma Aldrich, St. Louis, MO, USA).

Nojima I., Hosoda R., Toda Y., Saito Y., Ueda N., Horimoto K., Iwahara N., Horio Y, & Kuno A. (2022). Downregulation of IGFBP5 contributes to replicative senescence via ERK2 activation in mouse embryonic fibroblasts. Aging (Albany NY), 14(7), 2966-2988.

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Western Blot Analysis of Akt Phosphorylation

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Protein samples were diluted with Laemmli buffer (Bio‐Rad Laboratories) to 1 μg/μL and heat‐denatured for 4 min at 96°C. Samples (10 μg/lane) were loaded on a 12.5% polyacrylamide gel. The separated proteins were then transferred from the gel to a nitrocellulose membrane (GE Healthcare Inc., Piscataway, NJ) for 1 h at room temperature, and blocked for 1 h at room temperature in 3% blocking reagent (GE Healthcare Inc.) in tris buffered saline (TBS) buffer with 0.1% Tween 20 (TBS‐T). Membranes were incubated overnight at 4°C with rabbit monoclonal anti‐Akt (1:1000) or anti‐phospho‐Akt (pAkt) (ser473) (1:2000) antibodies (both from Cell Signaling Technology Inc, Danvers, MA) in TBS buffer. The subsequent incubation with secondary antibody (anti‐rabbit IgG) conjugated with horseradish peroxidase (1:15000) (GE Healthcare Inc.) was performed at room temperature for 1 h. The signals were developed using enhanced chemiluminescence (ECL) prime reagent (GE Healthcare Inc.) and imaged (Lumi‐Imager F1 LumiImager, Roche Mannheim Boehringer, Mannheim, Germany). The band intensities were determined using ImageJ Software (NIH).

Ichinose T., Lesmana R., Yamamoto A., Kobayashi T., Shitara H., Shimoyama D., Takatsuru Y., Iwasaki T., Shimokawa N., Takagishi K, & Koibuchi N. (2014). Possible involvement of IGF‐1 signaling on compensatory growth of the infraspinatus muscle induced by the supraspinatus tendon detachment of rat shoulder. Physiological Reports, 2(1), e00197.

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Immunohistochemistry and Western Blot Antibodies

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The following primary antibodies and dilutions were used for immunohistochemistry (IHC) staining and western blotting: rabbit polyclonal anti-UTX (1:1,000; Millipore, #ABE409); mouse monoclonal anti-SOX2 (1:500; R & D, #MAB2018); rabbit anti-PAX6 (1:1,000; Millipore, #AB2237); rabbit anti-TBR2 (1:1,000; Abcam, #AB23345); mouse monoclonal anti-β-ACTIN (1:20,000; Proteintech, #60008-1-Ig); rat monoclonal anti-BrdU (1:1,000; Abcam, #AB6362); rabbit monoclonal anti-Ki67 (1:1,000; Abcam, #AB15580); rabbit anti-PCNA (1:500; Santa Cruz Biotechnology, #SC7907), rabbit anti-TUJ1 (1:1,000; Sigma, #T2200); rabbit monoclonal anti-PTEN (1:1,000; Cell Signaling Technology, #9188); rabbit monoclonal anti-AKT (1:1,000; Cell Signaling, #4685); rabbit monoclonal anti-phospho-AKT (1:1,000; Cell Signaling, #3787); rabbit monoclonal anti-mTOR (1:1,000; Cell Signaling, #2983); rabbit monoclonal anti-phospho-mTOR (1:1,000; Cell Signaling, #5536); rabbit monoclonal anti-H3 (1:4,000; Cell Signaling, #4499S); rabbit polyclonal anti-trimethyl-histone H3 (Lys27) (1:2,000; Cell Signaling, #3377S); and rabbit anti-FLAG (1:1,000; Sigma, #7425).

Lei X, & Jiao J. (2018). UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling. Stem Cell Reports, 10(4), 1193-1207.

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Western Blot Analysis of Signaling Pathways

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Cells and tissue samples were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 1% NP40) supplemented with inhibitor cocktails of protease and phosphatase. Primary antibodies were mouse monoclonal anti-KRAS (Santa Cruz Biotechnology), rabbit polyclonal anti-ASNS (Abcam), rabbit monoclonal anti–pohospho-p44/42 kinase (Thr202/Tyr204) (Cell Signaling), rabbit monoclonal anti-p44/42 kinase (Cell Signaling), rabbit monoclonal anti–phospho-Akt (Ser473) (Cell Signaling), rabbit monoclonal anti-Akt (Cell Signaling), rabbit polyclonal anti–phospho-p70 S6 kinase (Thr389) (Cell Signaling), rabbit polyclonal anti-p70 S6 kinase (Cell Signaling), and mouse monoclonal anti–β-actin-peroxidase (Sigma-Aldrich).

Toda K., Kawada K., Iwamoto M., Inamoto S., Sasazuki T., Shirasawa S., Hasegawa S, & Sakai Y. (2016). Metabolic Alterations Caused by KRAS Mutations in Colorectal Cancer Contribute to Cell Adaptation to Glutamine Depletion by Upregulation of Asparagine Synthetase. Neoplasia (New York, N.Y.), 18(11), 654-665.

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Western Blot Analysis of Signaling Pathways

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Cells grown and treated as indicated were collected, and the total protein was extracted. The following primary antibodies were used: rabbit monoclonal anti-phosphorylated IGF-1R (Tyr1131), rabbit monoclonal anti-IGF-1R, rabbit monoclonal anti-Akt, rabbit monoclonal anti-phosphorylated Akt (Ser473), rabbit monoclonal anti-phosphorylated mTOR (Ser2448), rabbit monoclonal anti-mTOR, rabbit monoclonal anti-p70s6k, or rabbit monoclonal anti-phosphorylated p70s6k (Thr389), rabbit monoclonal anti-S6, or rabbit monoclonal anti-phosphorylated S6 (Ser240/244), rabbit monoclonal anti-AMPK, or rabbit monoclonal anti-phosphorylated AMPK (Thr172) (all from Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat-anti-rabbit antibody (Thermo Scientific) was used as a secondary antibody. The control for equal protein loading was assessed with an anti-β-actin antibody (Cell Signaling Technology, Inc.).

Li L., Wang Y., Peng T., Zhang K., Lin C., Han R., Lu C, & He Y. (2016). Metformin restores crizotinib sensitivity in crizotinib-resistant human lung cancer cells through inhibition of IGF1-R signaling pathway. Oncotarget, 7(23), 34442-34452.

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Protein Quantification and Western Blot Analysis

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Cells were lysed in buffer with 50 mM Tris–HCl pH 8, with 1% NP-40 (IgepalAC-630) 150 mM NaCl, 5 mM EDTA and fresh protease inhibitors. Extracts were sonicated for 10 s and centrifuged at 12000× rpm for 10 min to remove cell debris. Protein concentrations were determined by colorimetric assay (Bio-Rad). Western blotting was performed using the following primary antibodies: mouse monoclonal anti-Nucleolin (Abcam # ab13541), rabbit monoclonal anti-PTEN (Cell Signaling # 9552), rabbit monoclonal anti-AKT (Cell Signaling # 4685), rabbit monoclonal anti-P-AKT serin473 (Cell Signaling #4058). Secondary antibodies used were goat anti-mouse and goat anti-rabbit, conjugated to horseradish peroxidase (Amersham Biosciences,Piscataway, NJ). Immunostained bands were detected by chemiluminescent method (Pierce, Rockford, IL).

Vahabi M., Pulito C., Sacconi A., Donzelli S., D’Andrea M., Manciocco V., Pellini R., Paci P., Sanguineti G., Strigari L., Spriano G., Muti P., Pandolfi P.P., Strano S., Safarian S., Ganci F, & Blandino G. (2019). miR-96-5p targets PTEN expression affecting radio-chemosensitivity of HNSCC cells. Journal of Experimental & Clinical Cancer Research : CR, 38, 141.

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Western Blot Analysis of Signaling Pathways

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Cells were lysed with SDS sample buffer (70 mM Tris-HCl, 3% SDS, and 10% glycerol) with inhibitor cocktails of protease and phosphatase. Primary antibodies were rabbit monoclonal anti-phospho-p44/42 kinase (Thr202/Tyr204: Cell Signaling Technology, Danvers, MA, USA), anti-p44/42 kinase (Cell Signaling Technology), rabbit monoclonal anti-phospho-Akt (Ser473:Cell Signaling Technology), rabbit monoclonal anti-Akt (Cell Signaling Technology), rabbit polyclonal anti-phospho-p70 S6 kinase (Thr389: Cell Signaling Technology), rabbit polyclonal anti-p70 S6 kinase (Cell Signaling Technology), and mouse monoclonal anti-β-actin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA). For stimulation experiments, cells were starved for 24 h with serum-free DMEM and then stimulated with 100 ng/ml of CCL5. Cell lysates were obtained after the indicated times stimulation and subjected to SDS-polyacrylamide gel electrophoresis, immunoblotted with the primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and then analyzed by a lumino image analyzer.

Nishikawa G., Kawada K., Nakagawa J., Toda K., Ogawa R., Inamoto S., Mizuno R., Itatani Y, & Sakai Y. (2019). Bone marrow-derived mesenchymal stem cells promote colorectal cancer progression via CCR5. Cell Death & Disease, 10(4), 264.

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Western Blot Analysis of GC Cell Proteins

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Human GC tissues or cultured cells were lysed using RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA). Total protein was separated using 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Roche Diagnostics, Basel, Switzerland). The membranes were incubated at 4 °C with primary antibodies that included mouse monoclonal anti-RASSF9 (1:1000; Cell Signaling Technology, USA), mouse monoclonal anti-p-AKT (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-AKT (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Cyclin D1 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-CDK2 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bcl-2 (1:2000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bax (1:2000; Cell Signaling Technology, USA), and mouse monoclonal anti-GAPDH (1:5000; Santa Cruz Biotechnology, CA, USA). The membranes were subsequently treated with ECL reagent (Pierce, USA) for chemiluminescence detection. The luminescent signal was measured by a CCD camera and recorded and quantified with Syngene GBox (Syngene, UK).

Liu W.L., Wang H.X., Shi C.X., Shi F.Y., Zhao L.Y., Zhao W, & Wang G.H. (2019). MicroRNA-1269 promotes cell proliferation via the AKT signaling pathway by targeting RASSF9 in human gastric cancer. Cancer Cell International, 19, 308.

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Cell Culture and Antibody Preparation Protocol

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LNCaP cells were maintained in RPMI media (ATCC), containing 10% fetal bovine serum (Atlanta Biologicals) and penicillin-streptomycin (100 IU/ml, Mediatech). BHK-21 cells were maintained in DMEM (Sigma) containing 10% fetal bovine serum (Atlanta Biologicals), penicillin-streptomycin (100 IU/ml, Mediatech), L-Glutamine (2nM, Mediatech) and 1% Non-Essential Amino acids (Invitrogen). Primary antibodies used were as follows: rabbit monoclonal anti-AR (catalog #5153), rabbit monoclonal anti-PSA (catalog #5365), rabbit monoclonal anti-Akt (catalog #4685), rabbit monoclonal anti-phospho-Akt (catalog #4060), rabbit polyclonal anti-FLAG (catalog #2368) (Cell Signaling Technologies), mouse monoclonal anti-HIF-1α (catalog #610958, BD Biosciences), mouse polyclonal anti-α-tubulin (catalog #T6074, Sigma) and rabbit polyclonal anti-μNS (Qin et al., 2009 (link)). Secondary antibodies used for immunoblot experiments were alkaline phosphatase (AP)-conjugated goat anti-mouse (catalog #1706520) or anti-rabbit IgG (catalog #170-6518, Bio-Rad). Proteasome inhibitor, MG132 (Enzo Life Sciences), was used at a final concentration of 10 μM.

Gupta-Saraf P., Meseke T, & Miller C.L. (2015). Downregulation of key regulatory proteins in androgen dependent prostate tumor cells by oncolytic reovirus. Virology, 485, 153-161.

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Protein Expression Analysis of Cellular Pathways

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Cells grown and treated as indicated were collected and total protein was extracted. The following primary antibodies were used: rabbit monoclonal anti-α-actin (Abcam), monoclonal anti-COL1A1 (Millipore), rabbit monoclonal anti-phosphorylated SMAD2, rabbit monoclonal anti-phosphorylated SMAD3, rabbit monoclonal anti- SMAD2/3, rabbit monoclonal anti-ERK1/2, or rabbit monoclonal anti-phosphorylated ERK1/2, rabbit monoclonal anti-Akt, rabbit monoclonal anti-phosphorylated Akt, rabbit monoclonal anti-STAT3, or rabbit monoclonal anti-phosphorylated STAT3 (all from Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat-anti-rabbit antibody (Thermo Scientific) was used as a secondary antibody. The control for equal protein loading was assessed with an anti-β-actin antibody (Cell Signaling Technology, Inc.).

Li L., Huang W., Li K., Zhang K., Lin C., Han R., Lu C., Wang Y., Chen H., Sun F, & He Y. (2015). Metformin attenuates gefitinib-induced exacerbation of pulmonary fibrosis by inhibition of TGF-β signaling pathway. Oncotarget, 6(41), 43605-43619.

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