The following primary and secondary antibodies were used: anti-sarcomeric α-actinin (EA-53) and anti-Flag (F7425) (Sigma-Aldrich); anti-mouse lgG (sc-2025), anti-rabbit lgG (sc-2027), and β-actin (sc-47778) (Santa Cruz Biotechnology); anti-β5i (ab3329), anti-Myc (ab9106), VeriBlot for IP Detection Reagents (HRP) (ab131366), and anti-β5i (ab180606) (Abcam); anti-BNP (13299-1-AP) (Proteintech); anti-ATG5 (12994), anti–Beclin-1 (3495), anti-ATG7 (8558), anti-ATG12 (4180), anti-p62 (8025), anti-p53 (2524), anti-LC3B (3868), anti–phospho-AKT (9271), anti–phospho-AKT (9272), anti-ERK1/2 (9102), anti–phospho-ERK1/2 (9101), horseradish peroxidase–linked anti-mouse IgG (7074S), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118) (Cell Signaling Technology); and anti-ubiquitin (10201-2-AP) (Proteintech). The following reagents were used: Ang II (Aladdin); phenylephrine (Selleck); chloroquine, cycloheximide, and MG132 (Sigma-Aldrich); cell-based proteasome assay (Promega); and human PSMB8 enzyme-linked immunosorbent assay (ELISA) assay kit (Cloud-Clone).
Anti phospho akt
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, Germany, China, Japan, Macao
Anti-phospho-Akt is a laboratory research reagent that specifically detects the phosphorylated form of the protein kinase Akt (also known as protein kinase B). Akt is a key regulator of cellular processes such as survival, growth, and metabolism. This antibody can be used to measure the activation status of Akt in biological samples through techniques like Western blotting or immunohistochemistry.
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Lab products found in correlation
Anti akt, by Cell Signaling Technology (282 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (77 mentions)
Anti phospho erk, by Cell Signaling Technology (72 mentions)
Anti erk1 2, by Cell Signaling Technology (59 mentions)
Anti erk, by Cell Signaling Technology (55 mentions)
Anti β actin, by Cell Signaling Technology (50 mentions)
Anti phospho p38, by Cell Signaling Technology (42 mentions)
Anti phospho mtor, by Cell Signaling Technology (40 mentions)
Anti phospho jnk, by Cell Signaling Technology (39 mentions)
Anti β actin, by Merck Group (37 mentions)
Anti p38, by Cell Signaling Technology (34 mentions)
Anti mtor, by Cell Signaling Technology (34 mentions)
Anti gapdh, by Cell Signaling Technology (30 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (30 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (29 mentions)
Anti phospho stat3, by Cell Signaling Technology (29 mentions)
Anti jnk, by Cell Signaling Technology (25 mentions)
Anti total akt, by Cell Signaling Technology (25 mentions)
Anti bcl 2, by Cell Signaling Technology (24 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (24 mentions)
493 protocols using anti phospho akt
1
Antibody Profiling for Cardiac Protein Analysis
2
Quantitative Protein Analysis via Western Blot
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DHTDK1 antibody (Proteintech, 1:1000), β-Actin antibody (Proteintech, 1:5000), Total OXPHOS antibody (Abcam, 1:1000, this OXPHOS antibody contains five mouse mAbs, one each against Complex I NDUFB8, Complex II SDHB, Complex III UQCRC2, Complex IV MTCO1, and Complex V ATP5A), COX IV antibody (Cell Signaling, 1:1000), anti-phospho AKT (Cell Signaling, Ser473, 1:1000), anti-phospho AKT (Cell Signaling, Thr 308,1:1000), anti-AKT (Cell Signaling, 1:1000), anti-phospho ERK1/2 (Cell Signaling, 1:2000), anti-ERK1/2 (Cell Signaling, 1:1000), anti-phospho p38 (Cell Signaling, 1:2000), anti-p38 (Cell Signaling, 1:1000), anti-phospho JNK (Cell Signaling, 1:1000) and anti-JNK (Cell Signaling, 1:1000) were diluted in 5% milk, Tris-buffered saline TBS, and 0.1% Tween 20. Secondary antibodies (Sigma, Anti-Rabbit IgG, A3687; Sigma, Anti-Mouse IgG, A3562) were then used at 1:20000 dilution. Total protein was quantified prior to loading, and equal amounts of protein (30µg) loaded per lane. Bands were imaged by fluorescence, and relative protein abundance estimated using ImageJ.
3
Antibody Panel for ErbB Receptor Signaling
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Anti-neu/HER2 (ErbB2) antibody was from Invitrogen. Anti-ErbB2, anti-ErbB3, anti-EGFR, anti-caveolin-1, anti-phospho-ERK(Tyr204), anti-ERK, anti-phospho-Stat3(Tyr705), and anti-NDRG1 antibodies were from Santa Cruz Biotechnology INC. Anti-phospho-EGFR(Tyr1068) and anti-phospho-ErbB2(Tyr1248) antibodies were from Abcam. Anti-α-tubulin, anti-β-actin, and anti-cholera toxin B subunit antibodies were from Sigma Aldrich. Anti-4EBP1, anti-AMPKα, anti-acetyl-CoA carboxylase, anti-S6 ribosomal protein, anti-p70 S6 kinase, anti-Akt, anti-phospho-4EBP1(Thr37/46), anti-phospho-Akt(Thr308), anti-phospho-Akt(Ser473), anti-phospho-acetyl-CoA carboxylase(Ser79), anti-phospho-AMPKα(Thr172), anti-phospho-p70 S6 kinase(Thr 389), and anti-phospho-S6 Ribosomal protein(Ser240/244) antibodies were from Cell Signaling. Anti-Sestrin2 and anti-REDD1 antibodies were from Proteintech. Cholera toxin subunit B (CT-B) Conjugates were from Molecular Probes. CyTM3 Conjugate was from Jackson ImmunoResarch Lab. EGF was from Perprotech. MEDICA [α,α′-tetramethyl hexadecanedioic acid, HOOC-C(CH3)2-(CH2)12-C(CH3)2-COOH] was synthesized as previously described [17 (link), 26 ].
4
Western Blot Analysis of Cerebral Proteins
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Cerebral homogenates (50 μg) were electrophoresed onto sodium dodecyl sulfate gel and proteins were transferred to a PVDF membrane. Blots were blocked and probed with the following primary antibodies: anti‐glial fibrillary acidic protein (GFAP) (1:2000, monoclonal; Boehringer Mannheim GmbH), anti‐PKCα (1:1000, monoclonal; sc‐8393; Santa Cruz Biotechnology), anti‐phospho (p)‐AKT (1:1000, polyclonal; #4060; Cell Signaling Technology), anti‐AKT (1:1000, monoclonal; sc‐5298; Santa Cruz Biotechnology), anti‐ERK1/2 (1:1000, monoclonal; sc‐514302; Santa Cruz Biotechnology), and anti‐p‐ERK1 (1:1000, monoclonal; sc‐7383; Santa Cruz Biotechnology). β‐Actin monoclonal antibody (1:4000; sc‐8432; Santa Cruz Biotechnology) was used as control. Image J 1.45 software (https://imagej.nih.gov/ij/ ) was used for quantitative blot analyses.
5
Western Blot Antibody Validation
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Anti-β-actin antibody (sc-47778, monoclonal, raised in mouse, 1:10,000) was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), MTT, Hoechst 33258 and RNaseA were all purchased from Sigma-Aldrich. Anti-AKT (#9272, polyclonal, raised in rabbit, 1:1,000), anti-phospho (p)-AKT (ser473; #4058, monoclonal, raised in rabbit, 1:1,000), anti-human epidermal growth factor receptor 2 (Her2; #2165, monoclonal, raised in rabbit, 1:1,000), anti-epidermal growth factor receptor (EGFR; sc-03, polyclonal, raised in rabbit, 1:500), anti-c-Raf (#9422, polyclonal, raised in rabbit, 1:1,000), anti-B-cell lymphoma 2 (Bcl-2; #2876, polyclonal, raised in rabbit, 1:1,000) and anti-Bcl-2- associated X protein (Bax; #2772, polyclonal, raised in rabbit, 1:1,000) antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-cyclin-dependent kinase 4 (Cdk4; sc-260, polyclonal, raised in rabbit, 1:500), and anti-prostate-specific antigen (PSA; sc-7316, monoclonal, raised in mouse, 1:500), anti-AR (sc-7305, monoclonal, raised in mouse, 1:500), anti-Hsp70 (sc-24; monoclonal, raised in mouse, 1:500), Hsp90 (sc-69703, monoclonal, raised in mouse, 1:500) and NKX-3.1 (sc-15022, polyclonal, raised in goat, 1:500) antibodies were purchased from Santa Cruz Biotechnology, Inc. The Annexin V fluorescein isothiocyanate (FITC) Apoptosis Detection kit was purchased from BD Pharmingen (San Diego, CA, USA).
6
Quantitative Protein Expression Analysis
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Total protein was extracted from Hep-2 cells using RIPA buffer (Beyotime Institute of Biotechnology). Total protein was quantified using bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) and 80 µg protein/lane was separated using SDS-PAGE (10% gels) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk dissolved in PBST for 2 h at room temperature and incubated with the following primary antibodies: Anti-Bcl-2 (15071; 1:1,000), anti-Bax (5023; 1:1,000), anti-caspase-3 (9662; 1:1,000), anti-PTEN (9559; 1:1,000), anti-AKT (9272; 1:1,000), anti-phospho (p)-AKT (4060; 1:1,000), anti-NF-κB (8242; 1:1,000), anti-survivin (2808; 1:1,000; all Cell Signaling Technology, Inc., Danvers, MA, USA), anti-β-actin (612657; 1:1,000; BD Biosciences, Franklin Lakes, NJ, USA) diluted in PBS and incubated at 4°C overnight. Membranes were then washed with Tris-buffered saline with Tween-20 (TBST) and incubated with goat anti-rabbit IgG secondary antibody (Alexa Fluor-conjugated; A32730; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and quantified using Odyssey software version 1.2 (LI-COR Biosciences). GAPDH was used as an internal control.
7
Muscle Protein Expression Analysis
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The peripheral parts of muscle specimens were immediately frozen in liquid nitrogen and stored at −80°C for biochemical studies. Western blotting was performed as previously described (Fujimura and Usuki, 2015 (link), 2017 (link)). Briefly, the samples were sonicated for 5 s in tissue lysis buffer (T-PER Mammalian Protein Extraction Reagent; Pierce Biotechnology, Rockford, USA) containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, St Louis, USA). The samples were centrifuged (14,000 g for 1 h), and the supernatants were collected. The protein content was determined using the DC Protein Assay Kit II (Bio-Rad Laboratories, Hercules, USA). The cell lysates (20 μg protein) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel (Tefco, Tokyo, Japan) and transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were then subjected to the following antibody probes: anti-MGF (Millipore, Billerica, USA); anti-IGF-I (Santa Cruz Biotechnology, CA, USA); anti-YAP1 (Novus Biologicals, Centennial, CO, USA); anti-phospho-YAP1 (Abcam, Cambridge, UK); anti-PAX7 (Cytoskeleton Incorporated, Denver, USA); anti-phospho-ERK, anti-ERK, anti-AKT, anti-phospho-AKT, anti-phospho-4EBP1, and anti-phospho-p70 S6 kinase (Cell Signaling Technologies); and anti-β-actin (Sigma-Aldrich).
8
Western Blot Analysis of Cell Signaling
9
Quantitative Protein Analysis in Cell Lines
10
Quantification of Phosphorylated Proteins
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Cells were resuspended to a concentration of 2 million/ml in RPMI with 0.2% FBS. We stimulated cells with 10 nM fMLP and quenched the reaction at the indicated time points by adding aliquots of the cell mixture to ice-cold 20% trichloroacetic acid (TCA) containing the phosphatase inhibitors 40 mM NaF and 20 mM β-glycerol phosphate (50020; Fluka, St. Gallen, Switzerland). The samples were spun at 20,000 × g for 15 min to pellet. The sample pellets were washed with 0.5% TCA and resuspended in Laemmli protein sample buffer (161-0737; Bio-Rad) containing 5% β-mercaptoethanol. Protein bands were separated by SDS–PAGE gel electrophoresis, transferred to nitrocellulose, blocked with Odyssey block, and incubated at 4°C overnight with 1:1000 dilutions of anti–phospho-PAK (2605S; Cell Signaling, Danvers, MA) and anti-Pak2 (4825S; Cell Signaling) or anti–phospho-Akt (4060S; Cell Signaling) and anti-Akt (40D4; Cell Signaling). The blot was developed with the fluorescent secondary antibodies, and protein bands were imaged using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
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