Alexa fluor 647 goat anti rabbit igg

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Alexa Fluor 647 goat anti-rabbit IgG is a secondary antibody conjugate used in various immunological and cell biology applications. It is designed to detect and visualize rabbit primary antibodies. The Alexa Fluor 647 fluorescent dye is covalently attached to the goat anti-rabbit IgG antibody, allowing for sensitive and specific detection of target proteins or cellular structures.

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104 protocols using alexa fluor 647 goat anti rabbit igg

Comprehensive Platelet Signaling Evaluation

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Phospho-PKA Substrate (RRXS*/T*) Rabbit mAb and phospho-VASP-Ser^157/239 antibodies were from Cell Signaling Technology (Danvers, USA). PDE3A antibodies were from the MRC Unit (Dundee University, Dundee, UK). Anti-β-Tubulin antibody was from Millipore (Nottingham, UK). BD Phosflow Lyse/Fix Buffer was from BD Biosciences (Oxford, UK). OxPC-E06 mAb was from Avanti Polar Lipids (Alabaster, USA). FITC-labeled Rat Anti-Mouse P-selectin (CD62P) and PE-labeled JON/A antibodies were from Emfret Analytics (Würzburg, Germany). Alexa-Fluor 647 Goat anti-Rabbit IgG, Alexa-Fluor 488 Succinimidyl Ester and Pacific Blue Succinimidyl Ester were from ThermoFisher Scientific (Waltham, USA). PAR-1 peptide was from Anaspec (Fremont, USA). Anti-CD36 Antibody (FA6-152) was from Abcam (Cambridge, UK). Phosphodiesterase Activity Assay Kit was from Enzo Life Sciences (Exeter, UK). cAMP Biotrack EIA was from GE Healthcare (Buckinghamshire, UK). Horm Collagen was from Nycomed (Munich, Germany). PGI₂ and Cholesterol Assay Kit were from Cayman Chemical (Cambridge, UK). Vena8 Endothelial+ biochips were from Cellix (Hertfordshire, UK). All other reagents were from Sigma-Aldrich (Dorset, UK).

Berger M., Raslan Z., Aburima A., Magwenzi S., Wraith K.S., Spurgeon B.E., Hindle M.S., Law R., Febbraio M, & Naseem K.M. (2020). Atherogenic lipid stress induces platelet hyperactivity through CD36-mediated hyposensitivity to prostacyclin: the role of phosphodiesterase 3A. Haematologica, 105(3), 808-819.

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Immunofluorescence Staining of Lck and Src Phosphorylation

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Rabbit anti-Lck mAb-PE (73A5) mAb, rabbit anti-pY505-Lck (#2751), and rabbit anti-pY416-Src (#2101) polyclonal Abs were from Cell Signaling Technology. Rabbit anti-Lck (NBP1-85804) was from Novus Biologicals; mouse anti-pY505-Lck mAb-PE was from BD Biosciences; rat anti-human CD45 (YAML 501.4) Ab was from Santa Cruz Biotechnology; mouse anti-human CD45-AF647 (HI30) mAb was from BioLegend. For FCM and 3D-SIM, Abs were as follows: AlexaFluor 647 goat anti-rabbit IgG, AlexaFluor 594 donkey anti-rat IgG, and AlexaFluor 488 goat anti-rabbit IgG (Thermo Fischer). A770041 (Axon Medichem), Sodium Orthovanadate (Vanadate) New England BioLabs (NEB), catalase, and hydrogen peroxide (30%) are from Sigma-Aldrich.

Porciello N., Cipria D., Masi G., Lanz A.L., Milanetti E., Grottesi A., Howie D., Cobbold S.P., Schermelleh L., He H.T., D’Abramo M., Destainville N., Acuto O, & Nika K. (2022). Role of the membrane anchor in the regulation of Lck activity. The Journal of Biological Chemistry, 298(12), 102663.

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Cardiac Differentiation Protein Analysis

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AIC- and VIC-CD151^high/low CMs were sorted and then seeded at 1 × 10⁵ cells/well on a fibronectin-coated 96-well plate (Corning). The cells were fixed after 5–7 d of culture for 20 min with 4% paraformaldehyde, permeabilized for 15 min with 0.1% TritonX100-PBS, and then blocked for 1 h at room temperature with 2% goat serum/0.1% TritonX100-PBS. The fixed cells were stained using anti-MLC-2A (Synaptic systems, 1:100) and anti-MLC-2V (Proteintech, 1:200) overnight at 4 °C. The following day, the cells were washed twice with PBS and stained using Alexa Fluor 647 goat anti-mouse IgG (Thermo Fisher Scientific, 1:500), Alexa Fluor 647 goat anti-rabbit IgG (Thermo Fisher Scientific, 1:500), or Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific, 1:500) as the secondary antibody for 1 h at 4 °C under dark conditions. The cells were washed twice with PBS and stained with Hoechst (DOJINDO) for 5 min at room temperature. Images were captured using a BZ-X710 (KEYENCE) with a 20× objective.

Nakanishi-Koakutsu M., Miki K., Naka Y., Sasaki M., Wakimizu T., Napier S.C., Okubo C., Narita M., Nishikawa M., Hata R., Chonabayashi K., Hotta A., Imahashi K., Nishimoto T, & Yoshida Y. (2024). CD151 expression marks atrial- and ventricular- differentiation from human induced pluripotent stem cells. Communications Biology, 7, 231.

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Cortactin and Giantin Immunofluorescence

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Rabbit antihuman cortactin antibody (H222) (1:200) was purchased from Cell Signaling (#3503), rabbit antihuman giantin antibody (Poly19243) (1:000) was purchased from BioLegend (#924302). Secondary antibody Alexa Fluor 647 goat anti‐rabbit IgG (1:1000) was purchased from Thermo Fisher (#A‐21244).

Weiß M.S., Trapani G., Long H, & Trappmann B. (2024). Matrix Resistance Toward Proteolytic Cleavage Controls Contractility‐Dependent Migration Modes During Angiogenic Sprouting. Advanced Science, 11(19), 2305947.

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Characterizing Myogenic Progenitor Cells

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Phase-contrast microscopy, immunofluorescence staining, and qPCR analysis were performed as in [12 (link)]. Cell counting for Hoechst (Sigma–Aldrich, St. Louis, USA, B-2883) and T (Abcam, Toronto, Canada, AB20680) nuclear staining was performed as in [129 (link)]. Primary antibodies against PAX7 (Developmental Studies Hybridoma Bank, Iowa City, USA, AB528428), MKI67 (KI67)(Abcam, AB15580), MYOD1 (Abcam, AB133627), and secondary Cy3 goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, USA, 111-165-003), AlexaFluor546 goat anti-mouse IgG1 (ThermoScientific, Waltham, USA, A21123), and AlexaFluor647 goat anti-rabbit IgG (ThermoScientific, A21244) antibodies were also used. The antibody staining for PAX7, MYOD1, and KI67 were conducted similar to [13 (link)], with the exceptions that permeabilization was carried out with 0.5% TritionX-100 and 100 mM glycine in Tris-buffered saline (TBS), blocking solution was 2% bovine serum albumin (BSA), 5% goat serum, and 0.1% Tween20 in TBS, and the stains were mounted with PermaFluor (ThermoScientific, TA-006-FM). CellProfiler 3.0 was used to analyze the PAX7, MYOD1, and KI67 staining.

Shelton M., Ritso M., Liu J., O’Neil D., Kocharyan A., Rudnicki M.A., Stanford W.L., Skerjanc I.S, & Blais A. (2019). Gene expression profiling of skeletal myogenesis in human embryonic stem cells reveals a potential cascade of transcription factors regulating stages of myogenesis, including quiescent/activated satellite cell-like gene expression. PLoS ONE, 14(9), e0222946.

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Immunostaining of Drosophila Larval Hearts

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Immunostaining on whole mount larvae or dissected larval hearts was performed as previously described (Yang and Xu, 2012 ). The primary antibodies used in this study included the following: anti-phospho-Smad3 (rabbit; Abcam, 52903), anti-phospho-Smad1/5/9 (rabbit; CST, 13820), anti-phospho-histone H3 (rabbit; Merck Millipore, 06570), anti-MHC (mouse; DSHB, MF20), anti-Twist (mouse; Santa Cruz, 81417), and anti-Snail (rabbit; Genetex, 125918). The secondary antibodies included the following: Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 555 goat anti-mouse IgG, and Alexa Fluor 647 goat anti-rabbit IgG from Thermo Fisher Scientific.

Peng Y., Wang W., Fang Y., Hu H., Chang N., Pang M., Hu Y.F., Li X., Long H., Xiong J.W, & Zhang R. (2021). Inhibition of TGF-β/Smad3 Signaling Disrupts Cardiomyocyte Cell Cycle Progression and Epithelial–Mesenchymal Transition-Like Response During Ventricle Regeneration. Frontiers in Cell and Developmental Biology, 9, 632372.

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Immunodetection of NHE8 Protein

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Liver tissue sections (4–5 μm thick) and cultured human hepatocyte cells (HepG2) were used to detect the expression of NHE8 protein. The NHE8 antibody was used at a dilution of 1:200.^{2 (link)} For tissue samples, a goat anti-rabbit IgG (dilution 1:500; catalog no. SK-4105; Vector Laboratories, Inc, Burlingame, CA) was used as the secondary antibody, and a 3,3′-diaminobenzidine detection kit (catalog no. SK-4105; Vector Laboratories, Inc, Burlingame, CA) was used for signal detection. For cell samples, Alexa Fluor 647 goat anti-rabbit IgG (dilution 1:400; catalog No. A27040, Thermo Fisher Scientific) was used as the secondary antibody. Stained tissue sections and cells were then observed under microscope (EVOS FL Auto, Thermo Fisher Scientific).

Tong H., Bernardazzi C., Curiel L., Xu H, & Ghishan F.K. (2022). The Expression of NHE8 in Liver and Its Role in Carbon Tetrachloride–Induced Liver Injury. Gastro hep advances, 2(2), 199-208.

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Visualizing Curli Subunit CsgA in Biofilms

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An immunofluorescence assay targeting CsgA, the major curli subunit, was performed as previously described (9 (link)). Biofilm cryosections were fixed in 4% PFA for 30 min at room temperature and blocked overnight in 5% BSA at 4°C. Sections were washed in PBS, incubated with rabbit anti-CsgA antibodies (GenScript) (1:1,000) at room temperature for 1 h, washed in PBS, and incubated with Alexa Fluor 647 goat anti-rabbit IgG (ThermoFisher) (1:1,000) at room temperature for 1 h. Slides were counterstained with SYTO 9 and imaged using confocal laser scanning microscopy (CLSM).

Beebout C.J., Eberly A.R., Werby S.H., Reasoner S.A., Brannon J.R., De S., Fitzgerald M.J., Huggins M.M., Clayton D.B., Cegelski L, & Hadjifrangiskou M. (2019). Respiratory Heterogeneity Shapes Biofilm Formation and Host Colonization in Uropathogenic Escherichia coli. mBio, 10(2), e02400-18.

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Podocyte Extracellular Matrix Analysis

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The mouse anti-human podocalyxin antibody (10 μg/mL) was purchased from R&D Systems (MAB1658). Rabbit anti-collagen IV antibody (1:100) was obtained from Abcam (ab6586). Mouse anti-integrin α_vβ₃ antibody (20 μg/mL) was purchased from Merck Chemicals (MAB1976Z). Rabbit anti-laminin (1:500), rabbit anti-laminin 411 (1:500), rabbit anti-laminin 511 (1:500), and mouse anti-integrin β1 (20 μg/mL) were prepared by the laboratory of Lydia Sorokin. The secondary antibodies Alexa Fluor 555 donkey anti-mouse IgG (1:200) and Alexa Fluor 647 goat anti-rabbit IgG (1:200) were purchased from Thermo Fisher (A-31570 and A-21244, respectively).

Liu J., Long H., Zeuschner D., Räder A.F., Polacheck W.J., Kessler H., Sorokin L, & Trappmann B. (2021). Synthetic extracellular matrices with tailored adhesiveness and degradability support lumen formation during angiogenic sprouting. Nature Communications, 12, 3402.

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Multicolor Immunofluorescence Imaging Protocol

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Primary Antibodies: anti-CENPA (MBL international 1:500, Code # D115-3), anti-CENPB primary antibody (Santa Cruz Biotechnology, CENPB Antibody (C-10) 1:50: sc-376392, pSer139-γ-H2AX antibody (Cell Signaling 1:400, Cat # 9718), BANF1/BAF antibody (abcam,1:100, EPR7668), MHCII DRB1 antibody (Abclonal, 1:100, Cat # A7685), Lamin B1 antibody (Proteintech, 1:1000, Cat # 66095-1-lg), cGAS antibody (Abclonal, 1:100, Cat# A8335), p(Ser396)-IRF antibody (Cell Signaling, 1:100, Cat # 29047), p(Ser536)-p65 antibody (Abclonal, 1:100, Cat # AP0123), MHCII DRB5 antibody (Abclonal, 1:100, Cat # A12726), anti-GAPDH antibody (Abcam, ab 9484), anti-Histone H3 (tri methyl K9) antibody (Abcam, ab8898), anti-CENPA antibody [3-19] (Abcam, ab13939).
Secondary Antibodies: Alexa Flour 594 rabbit anti-mouse IgG 1:1000, Thermo Fischer Scientific, Cat No A27027, Alexa Flour 488 rabbit anti-mouse IgG 1:1000, Thermo Fischer Scientific, Cat No A27023. Alexa fluor 647 Goat anti-rabbit IgG, ThermoFisher, 1:1000, Cat # A32733TR

Paul S., Kaplan M.H., Khanna D., McCourt P.M., Saha A.K., Tsou P.S., Anand M., Radecki A., Mourad M., Sawalha A.H., Markovitz D.M, & Contreras-Galindo R. (2022). Centromere defects, chromosome instability, and cGAS-STING activation in systemic sclerosis. Nature Communications, 13, 7074.

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