Alexa fluor 680 conjugated secondary antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 680-conjugated secondary antibodies are fluorescent-labeled antibodies used for detection and visualization in various immunoassay techniques. They are designed to bind to primary antibodies and emit fluorescence at a wavelength of 680 nm when excited.

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Lab products found in correlation

Odyssey system, by LI COR (4 mentions) Odyssey infrared imaging system, by LI COR (3 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Phosphatase inhibitor mixture, by Merck Group (2 mentions) Protease inhibitor mixture, by Merck Group (2 mentions) Pvdf membrane, by Merck Group (2 mentions) S6 ribosomal protein, by Cell Signaling Technology (1 mentions) Rabbit anti ha, by Merck Group (1 mentions) Mouse anti tubulin, by Merck Group (1 mentions) Mouse anti c myc 9e10 sc 40, by Santa Cruz Biotechnology (1 mentions) Anti ucp1 antibody, by Abcam (1 mentions) Ab23841, by Abcam (1 mentions) Dnmt3b, by Santa Cruz Biotechnology (1 mentions) Mitochondrial total oxphos protein antibody set, by Abcam (1 mentions) Ab152, by Merck Group (1 mentions) Ab23841, by Merck Group (1 mentions) Anti gapdh, by Thermo Fisher Scientific (1 mentions) Anti flag, by Thermo Fisher Scientific (1 mentions) Anti ha, by Novus Biologicals (1 mentions) Anti cleaved caspase 1, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Alexa Fluor-conjugated secondary antibodies (1427 citations) Alexa Fluor secondary antibodies (639 citations) Alexa-conjugated secondary antibodies (274 citations) Alexa Fluor 680 (241 citations) Alexa 680 (31 citations) Alexa Fluors (21 citations) Alexa Fluor 680 secondary antibody (8 citations) Alexa Fluor-conjugated secondary (4 citations)

15 protocols using alexa fluor 680 conjugated secondary antibody

Cell Signaling Pathway Analysis

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Tissue lysates were prepared for cell signaling analyses as previously described in Duchêne et al. (2008) (link). Lysates were first centrifuged at 1000 g for 30 min at 10°C and supernatants were then centrifuged at 31,000 g for 45 min at 10°C. Solubilized lysates (containing 40 μg of protein) were subjected to SDS-PAGE and Western blotting with the appropriate antibody. Antibodies raised against phospho-S6K1 [T389], phospho-S6 [S235/S236], and S6 ribosomal protein, were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-S6K1, and anti-vinculin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States), Upstate (Millipore, Paris, France), and Sigma Chemical Company (St. Louis, MO, United States).
After washing, membranes were incubated with a DyLight® 680-conjugated antibody. Alexa Fluor 680-conjugated secondary antibodies were obtained from Molecular Probes (Invitrogen, Carlsbad, CA, United States). Bands were visualized with Infrared Fluorescence using the Odyssey® Imaging System (LI-COR Inc. Biotechnology, Lincoln, NE) and quantified with the Odyssey imaging system software (Application software, version 1.2).

Métayer-Coustard S., Tesseraud S., Praud C., Royer D., Bordeau T., Coudert E., Cailleau-Audouin E., Godet E., Delaveau J., Le Bihan-Duval E, & Berri C. (2021). Early Growth and Protein-Energy Metabolism in Chicken Lines Divergently Selected on Ultimate pH. Frontiers in Physiology, 12, 643580.

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Immunoblotting Antibody Characterization

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Biotinylated mouse anti-FLAG (M2, 1:500, Sigma Aldrich), rabbit anti-HA (1:500, Sigma Aldrich) and rabbit anti-VP30 (Biedenkopf et al., 2016a) , rabbit anti-VP30 pSer29 antibody (1:100, (Lier et al., 2017) ), chicken anti-EBOV NP (1:1000, (Pauly et al., 2011) ), mouse anti-c-Myc (9E10 sc-40, 1:1000, Santa Cruz Biotechnology), mouse anti-B56a (610615, 1:3000, BD Biosciences), rabbit anti-PP2A-A scaffold subunit (2041, 1:1000, Cell Signaling Technology), rabbit anti-GFP (ab290, 1:10000, Abcam), goat anti-GFP (600-101-215, 1:1000, Rockland), mouse anti-PP2A-C catalytic subunit (05-421,1:2000, Millipore), rabbit anti-PP1g (A300-906A, 1:1000, Bethyl), mouse anti-B55a (5689S, 1:1000, Cell Signaling Technology), rabbit anti-B55b (HPA042122, 1:1000, Atlas Antibodies), mouse anti-Tubulin (1:1000, Sigma-Aldrich). Alexa Fluor 680-conjugated secondary antibodies were purchased from Invitrogen Molecular Probes (Thermo Fisher Scientific), IRDye 800-conjugated antibodies came from Rockland (all diluted 1:5000). Detection of antibodies was performed using the Li-Cor Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA).

Kruse T., Biedenkopf N., Hertz E.P.T., Dietzel E., Stalmann G., López-Méndez B., Davey N.E., Nilsson J, & Becker S. (2018). The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30. Molecular cell, 69(1).

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Recombinant Protein Detection Immunoblot

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Cell lines expressing the recombinant proteins or bacteria expressing the peptide-infused proteins were subjected to 4% to 20% SDS-PAGE (GenScript catalog number M42015C) for protein separation, transferred onto a nitrocellulose membranes, and blocked with 5% skimmed milk at 4°C overnight. The membranes were then probed with MAb 22.9-1 at 37°C for 1 h, washed three times with PBST, incubated with anti-mouse Alexa Fluor 680-conjugated secondary antibodies (catalog number A10038, Thermo) or HRP-conjugated goat anti-mouse IgG for 1 h at 37°C, and then washed three times with PBST. Protein bands were detected with the Li-Cor Odyssey system (Li-Cor Biosciences, Lincoln, NE, USA) or developed with the enhanced chemiluminescence substrate.

Hua R.H., Zhang S.J., Niu B., Ge J.Y., Lan T, & Bu Z.G. (2023). A Novel Conserved Linear Neutralizing Epitope on the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein. Microbiology Spectrum, 11(4), e01190-23.

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Immunoblotting for Protein Detection in Fat Tissues

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Immunoblotting for protein detection was conducted as we described [27 (link),32 (link)]. Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). Tissue lysates were resolved by SDS-PAGE gels. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (ThermoFisher Scientific). The blots were developed with a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). Primary antibodies used were as follows: Anti-UCP1 antibody (1:500, ab23841, Abcam, Cambridge, MA, USA); Anti-α-Tubulin antibody (1:1000, ABCENT4777, Advanced BioChemicals, Lawrenceville, GA, USA); Mitochondrial total OXPHOS protein antibody set (Abcam,ab110413); and Anti-pHSL (4126s, Cell Signaling Technology, Danvers, MA, USA); DNMT3b (sc-393845, Santa Cruz, Dallas, TX, USA, sc-393845); HSL (Cell Signaling, 4107s).

Wang S., Cao Q., Cui X., Jing J., Li F., Shi H., Xue B, & Shi H. (2021). Dnmt3b Deficiency in Myf5+-Brown Fat Precursor Cells Promotes Obesity in Female Mice. Biomolecules, 11(8), 1087.

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Western Blot Analysis of Adipose Tissue

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Adipose tissue was homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with a protease inhibitor mixture and phosphatase inhibitor mixture (Sigma, St. Louis, MO, USA). Tissue homogenates were separated by SDS-PAGE, which were transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), followed by blocking, washing, and incubating with primary antibodies and Alexa Fluor 680-conjugated secondary antibodies (ThermoFisher Scientific, Waltham, MA, USA). The blots were developed with a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). The primary antibodies are UCP1 (1:500, Abcam, ab23841, Cambridge, MA, USA), TH (1:1000, Millipore, AB152, Burlington, MA, USA), phospho-HSL (pHSL) (1:1000, Cell Signaling, 4126, Danvers, MA, USA), CD36 (1:1000, Abnova, PAB 12463, Walnut, CA, USA) and α-Tubulin (1:1000, Advanced BioChemicals, ABCENT4777, Lawrenceville, GA, USA).

Cao Q., Wang S., Wang H., Cui X., Jing J., Yu L., Shi H, & Xue B. (2021). Fatty Acids Rescue the Thermogenic Function of Sympathetically Denervated Brown Fat. Biomolecules, 11(10), 1428.

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ASFV Protein Detection by Western Blot

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Lysates of ASFV-infected cells or bacterial-expressed peptide fusion proteins were separated by 4 %–20 % SDS–PAGE (GenScript, M42015C), transferred onto nitrocellulose membranes, and blocked with 5 % skimmed milk at 4 °C overnight. Then, the membranes were probed with K205R-specific monoclonal antibody or ASFV-infected pig serum at 37 °C for 1 h, washed three times with PBS plus 0.5% Tween-20 (PBST), and incubated with anti-mouse Alexa Fluor 680-conjugated secondary antibodies (Thermo, A10038) or horseradish peroxidase (HRP)-conjugated goat anti-pig IgG for 1 h at 37 °C, followed by three washes with PBST. Protein bands were detected using the Li-Cor Odyssey system (Li-Cor Biosciences, Lincoln, NE, USA) or developed with ECL substrate.

Zhang S.J., Liu J., Niu B., Zhu Y.M., Zhao D.M., Chen W.Y., Liu R.Q., Bu Z.G, & Hua R.H. (2023). Comprehensive mapping of antigenic linear B-cell epitopes on K205R protein of African swine fever virus with monoclonal antibodies. Virus Research, 328, 199085.

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Recombinant Protein Detection Immunoblot

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Immunoblotting Technique for Protein Analysis

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Immunoblotting was performed as previously described (Liu et al., 2015 (link)). In brief, cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% Na-deoxycholate acid) and then denatured using SDS sample buffer. Cell lysates were subsequently subjected to 10% SDS-PAGE followed by membrane transfer (Immobilon-P and Towbin transfer buffer, pH 8.3; EMD Millipore). Immunoblots on the membrane were blocked with 5% nonfat milk dissolved in Tris-buffered saline with Tween (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween) and then probed with primary antibodies diluted in Tris-buffered saline with Tween. Primary antibodies were visualized using Alexa Fluor 680–conjugated secondary antibodies (Thermo Fisher Scientific) together with the LI-COR imaging system (LI-COR Biosciences).

Liu C, & Mao Y. (2016). Diaphanous formin mDia2 regulates CENP-A levels at centromeres. The Journal of Cell Biology, 213(4), 415-424.

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Western Blotting of Protein Targets

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The liver tissues and cell lines were lysed using radioimmunoprecipitation assay (RIPA) buffer (#P1003B, Beyotime). Approximately 30 μg of total protein was added to 8% SDS PAGE gels using Bio-Rad equipment (Shanghai, China). After separation, the proteins were transferred to PVDF membranes (#ISEQ00010, Millipore, Massachusetts, USA). The protein-loaded PVDF membranes were blocked with 5% skim milk (#A600669, Sangon), immersed in solutions of the primary antibodies at 4 °C overnight, and then incubated with Alexa Fluor 680-conjugated secondary antibodies (Thermo Fisher Scientific). Tubulin-α or GAPDH was used as an internal control. The used primary antibodies were listed (anti-NLRP3, #IMG-6668A, Novus Biologicals, Colorado, USA; anti-MARCH7, #PA5-54572, Sigma Aldrich; anti-Cleaved caspase-1, #PA5-99390, Thermo Fisher Scientific; anti-Matured IL-1β, #AF401, R&D Systems, City of Emeryville, USA; anti-GSDMD-N, #GSDMD antibody Abcam EPR 19828, Abcam, Cambridge, UK; anti-Tubulin-α, #NB100-690, Novus Biologicals; anti-GAPDH, #MA1-16757, Thermo Fisher Scientific; anti-actin-β, #NB600-501, Novus Biologicals; anti-HA, #NB600-363, Novus Biologicals; anti-Flag, #MA1-91878, Thermo Fisher Scientific; anti-Ubiquitin, #NB300-130, Novus Biologicals; anti-GST, #13-6700, Thermo Fisher Scientific; anti-His, #NBP2-61482, Novus Biologicals; anti-Myc, #2276, Cell Signaling Technology, Boston, USA).

Chen T., Meng Y., Zhou Z., Li H., Wan L., Kang A., Guo W., Ren K., Song X., Chen Y, & Zhao W. (2023). GAS5 protects against nonalcoholic fatty liver disease via miR-28a-5p/MARCH7/NLRP3 axis-mediated pyroptosis. Cell death and differentiation, 30(7).

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Whole-cell Western Blotting Protocol

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Whole-cell extract preparation and Western blotting were performed as described in reference (Du et al., 2013 (link)). Membrane were probed with phosphor-specific Rb (Ser⁷⁹⁵), Phospho-specific ERK (T²⁰²/Y²⁰⁴), Phospho-specific S6 (S^235/236), Rab 11 primary antibodies (Cell Signaling Technology, Denver, MA), followed by AlexaFluor® 680-conjugated secondary antibodies (Invitrogen, Carlsbad, CA). Images were acquired using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).

Du Z., Treiber D., McCarter J.D., Fomina-Yadlin D., Saleem R.A., McCoy R.E., Zhang Y., Tharmalingam T., Leith M., Follstad B.D., Dell B., Grisim B., Zupke C., Heath C., Morris A.E, & Reddy P. (2014). Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures. Biotechnology and Bioengineering, 112(1), 141-155.

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