Alexa fluor 680 goat anti mouse igg, by Thermo Fisher Scientific - Product details

1

Western Blot Analysis of Cellular Proteins

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Lysates were resolved on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with anti-vinculin (Sigma, 1:10000), anti-ATP6V1A (Abnova, 1:1000), anti-ATP6V0a3 (Novus, Centennial, CO; 1:1000), anti-FLAG (Sigma, 1:2000), anti-GFP (Cell Signaling, 1:1000), anti-T7 (Novagen, 1:10000), anti-tubulin (Sigma, 1:10000), anti-Rac1 (BD Transduction Laboratories, 1:1000), anti-AKT (Cell Signaling, 1:1000), anti-pAKT (S473) (Cell Signaling, 1:500), anti-ERK2 (EMD Millipore, 1:2000), anti-pERK1/2 (Cell Signaling, 1:1000), anti-SLC4A7 (Santa Cruz, 1:200), or anti-KRas (Santa Cruz, 1:500) primary antibodies followed by Alexa Fluor 680 goat anti-mouse IgG (Life Technologies, 1:10000) or IRDye 800CW goat anti-rabbit IgG (Li-Cor, 1:10000) secondary antibodies. Blots were analyzed using an Odyssey Classic imager (Li-Cor).

Ramirez C., Hauser A.D., Vucic E.A, & Bar-Sagi D. (2019). Plasma membrane v-ATPase controls oncogenic Ras-induced macropinocytosis. Nature, 576(7787), 477-481.

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2

Western Blot Analysis of Cell Proteins

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Whole cell extracts of cells were prepared and resolved as previously described^{58 (link)}. Primary antibodies used include Cx43 (Abcam, ab11370), PR (Dako, PgR-1294), c-Kit (Santa Cruz Biotechnology, sc-168) or c-Kit (R&D systems, AF1356), ERα (SP1, Thermofisher, RM-9101) α-tubulin (Sigma-Aldrich, T5168), GAPDH (Cell Signaling, 2118S). Secondary antibodies included were Alexa-fluor 680 Goat-Anti-Mouse IgG (A21058; Lifetechnologies, Thermofisher, USA) and Alexa-fluor 680 Goat-Anti-Rabbit IgG (A21109; Lifetechnologies, Thermofisher, USA). Immunoblots were imaged using the Odyssey Infrared Imaging System (Li-Cor Biosciences). Experiments were repeated at least twice.

Harrell J.C., Shroka T.M, & Jacobsen B.M. (2017). Estrogen induces c-Kit and an aggressive phenotype in a model of invasive lobular breast cancer. Oncogenesis, 6(11), 396.

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3

Western Blot Analysis of Cellular Signaling Proteins

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Protein was extracted, quantified, and probed with antibody, as previously described.^{69 (link)} Whole cell lysates were extracted using RIPA buffer (Cell Signaling). Nuclear and cytoplasmic lysates were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit, according to the manufacturer (Thermo Scientific). Antibodies included AKT (Cell Signaling, Danvers, MA), phospho-AKT (S473) (Cell Signaling), CXCR4 (Abcam, Cambridge, MA), phospho-CXCR4 (S339) (Abcam), eEF2 (Cell Signaling), GRK4-6 (EMD Millipore, Billerica, MA), GRK5 (C-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), KCNA1 (Abcam), Lamin B1 (Cell Signaling), NPR3 (Abcam), WIP1 (Bethyl, Montgomery, TX), and β-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies Alexa Fluor 680 goat anti-mouse IgG (Life Technologies) or IRDye 800 goat anti-rabbit IgG (Rockland, Gilbertsville, PA) were used at a dilution of 1:5,000. Immunoblots were imaged and quantified, as previously described.^{27 (link)}

Buss M.C., Remke M., Lee J., Gandhi K., Schniederjan M.J., Kool M., Northcott P.A., Pfister S.M., Taylor M.D, & Castellino R.C. (2014). The WIP1 Oncogene Promotes Progression and Invasion of Aggressive Medulloblastoma Variants. Oncogene, 34(9), 1126-1140.

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4

Quantification of A3B Protein Expression

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Propagation of 293T cells, transfection, preparation of cell extracts, and determination of protein concentration were performed as described previously^{44 (link),92 (link)}. The relative expression levels of A3B were determined by Western blot analysis of cell extracts (10 μg of total protein for each sample), using the Western Breeze chemiluminescent Western blot kit (Life Technologies, Grand Island, NY). The primary antibodies were anti-HA (Cell Signaling Technologies, Danvers, MA) for detection of all A3B proteins and anti-tubulin (Abcam, Cambridge, MA) for the loading control. The secondary antibodies, AlexaFluor 488 goat anti-rabbit IgG and AlexaFluor 680 goat anti-mouse IgG, were obtained from Life Technologies.

Byeon I.J., Byeon C.H., Wu T., Mitra M., Singer D., Levin J.G, & Gronenborn A.M. (2016). Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity. Biochemistry, 55(21), 2944-2959.

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5

Western Blot Analysis of Firefly Luciferase

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5 μg of whole tissue lysates were resolved in 4–12% Bis–Tris denaturing NuPAGE gels (Life Technologies) and transferred to nitrocellulose membranes. Membranes were cut at the 50 kDa marker, blocked for 1 h at RT in blocking buffer (3% BSA; 1×TBS, 0.5% Tween 20), and incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The top half of the membrane was probed with a rabbit polyclonal antibody to firefly luciferase (GTX125849, Genetex, Irvine, USA) diluted 1:1000 and the bottom half was probed with mouse monoclonal antibody to β-actin (A5441, Sigma-Aldrich, UK) diluted 1:5000. Membranes were washed, incubated with 1:5000 Alexa Fluor® 680 goat anti-rabbit IgG and Alexa Fluor® 680 goat anti-mouse IgG (Life Technologies) and imaged using the LI-COR Odyssey (LI-COR, Cambridge, UK) as previously described [14] (link).

Hegde V., Hickerson R.P., Nainamalai S., Campbell P.A., Smith F.J., McLean W.H, & Leslie Pedrioli D.M. (2014). In vivo gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation. Journal of Controlled Release, 196, 355-362.

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6

Detecting MUC2 Protein Expression

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Whole cell extracts of cells were prepared and resolved as previously described [26 (link)]. Primary antibody was MUC2 (Thermo MS-1729-P1) and loading control α-tubulin (Sigma-Aldrich, T5168). Secondary antibodies were Alexa-fluor 680 Goat-Anti-Mouse IgG and Alexa-fluor 680 Goat-Anti-Rabbit IgG (A21058 and A21109; Lifetechnologies, Thermofisher, USA). Immunoblots were imaged using the Odyssey Infrared Imaging System (Li-Cor Biosciences). Experiments were repeated at least twice.

Astashchanka A., Shroka T.M, & Jacobsen B.M. (2018). Mucin 2 (MUC2) modulates the aggressiveness of breast cancer. Breast cancer research and treatment, 173(2), 289-299.

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7

Western Blot Analysis of Cellular Signaling Proteins

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Protein was extracted, quantified, and probed with antibody, as previously described.^{69 (link)} Whole cell lysates were extracted using RIPA buffer (Cell Signaling). Nuclear and cytoplasmic lysates were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit, according to the manufacturer (Thermo Scientific). Antibodies included AKT (Cell Signaling, Danvers, MA), phospho-AKT (S473) (Cell Signaling), CXCR4 (Abcam, Cambridge, MA), phospho-CXCR4 (S339) (Abcam), eEF2 (Cell Signaling), GRK4-6 (EMD Millipore, Billerica, MA), GRK5 (C-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), KCNA1 (Abcam), Lamin B1 (Cell Signaling), NPR3 (Abcam), WIP1 (Bethyl, Montgomery, TX), and β-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies Alexa Fluor 680 goat anti-mouse IgG (Life Technologies) or IRDye 800 goat anti-rabbit IgG (Rockland, Gilbertsville, PA) were used at a dilution of 1:5,000. Immunoblots were imaged and quantified, as previously described.^{27 (link)}

Buss M.C., Remke M., Lee J., Gandhi K., Schniederjan M.J., Kool M., Northcott P.A., Pfister S.M., Taylor M.D, & Castellino R.C. (2014). The WIP1 Oncogene Promotes Progression and Invasion of Aggressive Medulloblastoma Variants. Oncogene, 34(9), 1126-1140.

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8

Immunoblotting Analysis of p53 Regulatory Proteins

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Protein was extracted, quantified, and probed with antibodies, as described.^{40 (link)} Whole cell lysates were extracted using RIPA buffer (Cell Signaling). Antibodies used included p53 (Cell Signaling, Danvers, MA), Mdm2 (EMD Millipore, Billerica, MA), WIP1 (Bethyl, Montgomery, TX), and β-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies Alexa Fluor 680 goat anti-mouse IgG (Life Technologies) or IRDye 800 goat anti-rabbit IgG (Rockland, Gilbertsville, PA) were used at a dilution of 1:5,000. Immunoblots were imaged as previously described.^{29 (link)}

Wen J., Lee J., Malhotra A., Nahta R., Arnold A.R., Buss M.C., Brown B.D., Maier C., Kenney A.M., Remke M., Ramaswamy V., Taylor M.D, & Castellino R.C. (2016). WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma. Oncogene, 35(42), 5552-5564.

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9

Western Blot Analysis of Cellular Proteins

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Lysates were resolved on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with anti-vinculin (Sigma, 1:10000), anti-ATP6V1A (Abnova, 1:1000), anti-ATP6V0a3 (Novus, Centennial, CO; 1:1000), anti-FLAG (Sigma, 1:2000), anti-GFP (Cell Signaling, 1:1000), anti-T7 (Novagen, 1:10000), anti-tubulin (Sigma, 1:10000), anti-Rac1 (BD Transduction Laboratories, 1:1000), anti-AKT (Cell Signaling, 1:1000), anti-pAKT (S473) (Cell Signaling, 1:500), anti-ERK2 (EMD Millipore, 1:2000), anti-pERK1/2 (Cell Signaling, 1:1000), anti-SLC4A7 (Santa Cruz, 1:200), or anti-KRas (Santa Cruz, 1:500) primary antibodies followed by Alexa Fluor 680 goat anti-mouse IgG (Life Technologies, 1:10000) or IRDye 800CW goat anti-rabbit IgG (Li-Cor, 1:10000) secondary antibodies. Blots were analyzed using an Odyssey Classic imager (Li-Cor).

Ramirez C., Hauser A.D., Vucic E.A, & Bar-Sagi D. (2019). Plasma membrane v-ATPase controls oncogenic Ras-induced macropinocytosis. Nature, 576(7787), 477-481.

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10

Immunoblotting Analysis of p53 Regulatory Proteins

Cited 1 time

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Protein was extracted, quantified, and probed with antibodies, as described.^{40 (link)} Whole cell lysates were extracted using RIPA buffer (Cell Signaling). Antibodies used included p53 (Cell Signaling, Danvers, MA), Mdm2 (EMD Millipore, Billerica, MA), WIP1 (Bethyl, Montgomery, TX), and β-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies Alexa Fluor 680 goat anti-mouse IgG (Life Technologies) or IRDye 800 goat anti-rabbit IgG (Rockland, Gilbertsville, PA) were used at a dilution of 1:5,000. Immunoblots were imaged as previously described.^{29 (link)}

Wen J., Lee J., Malhotra A., Nahta R., Arnold A.R., Buss M.C., Brown B.D., Maier C., Kenney A.M., Remke M., Ramaswamy V., Taylor M.D, & Castellino R.C. (2016). WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma. Oncogene, 35(42), 5552-5564.

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Alexa fluor 680 goat anti mouse igg

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42 protocols using alexa fluor 680 goat anti mouse igg

Western Blot Analysis of Cellular Proteins

Western Blot Analysis of Cell Proteins

Western Blot Analysis of Cellular Signaling Proteins

Quantification of A3B Protein Expression

Western Blot Analysis of Firefly Luciferase

Detecting MUC2 Protein Expression

Western Blot Analysis of Cellular Signaling Proteins

Immunoblotting Analysis of p53 Regulatory Proteins

Western Blot Analysis of Cellular Proteins

Immunoblotting Analysis of p53 Regulatory Proteins

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