The primary antibodies that were used for IFA staining are: Mouse anti-dsRNA (English & Scientific Consulting, Szirák, Hungary), Rabbit anti-dsRNA (Absolute antibody, Oxford, UK); Mouse anti-hnRNP K (Santa Cruz, California, USA); Goat anti-eIF3 (Santa Cruz, California, USA); Rabbit anti-G3BP2 (Bethyl Laboratories, Texas, USA). The corresponding secondary antibodies that were used for IFA staining are: Alexa Fluor 488-, Alexa Fluor 594-, and Alexa Fluor 647-conjugated secondary antibodies (Invitrogen, California, USA). The primary antibodies that were used for WB are: Rabbit anti-eIF4G (Bethyl Laboratories, Texas, USA); Mouse anti-tubulin (Sigma, Andalucia, Spain). The monoclonal rat anti-2A antibody was a kind gift from Malin Flodström-Tullberg. Respective IRdye680- or IRdye800-conjugated secondary antibodies (LiCOR, Lincoln, NE, USA) were used for detection.
Irdye800 conjugated secondary antibodies
Manufactured by LI COR
Sourced in United States, Germany
IRDye800-conjugated secondary antibodies are fluorescent-labeled antibodies designed for use in various immunoassays and imaging applications. These antibodies are conjugated with the near-infrared dye IRDye800, which provides high sensitivity and low background signal. The core function of these secondary antibodies is to bind and detect primary antibodies, enabling the visualization and quantification of target proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (7 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Odyssey application software v2, by LI COR (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Protease inhibitor cocktail tablet, by Roche (2 mentions)
Irdye 680, by LI COR (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rabbit anti g3bp2, by Fortis Life Sciences (1 mentions)
Mouse anti tubulin, by Merck Group (1 mentions)
Blocking buffer, by LI COR (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Beyotime (1 mentions)
α flag, by Merck Group (1 mentions)
α gfp, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
16 protocols using irdye800 conjugated secondary antibodies
1
Antibody Characterization for IFA and WB
2
Immunoblotting Protocol for Gamma-Herpesvirus Proteins
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Cells were collected, rinsed with ice-cold PBS and lysed in NP-40 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 5 mM EDTA) supplemented with a protease inhibitor cocktail (Roche), and sonicated for 3 times on ice. Whole cell lysates were denatured and resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membrane and blocked in PBS-T containing 3% non-fat milk for one hour at room temperature. Membrane was incubated with primary antibodies overnight at 4°C. Blots were washed extensively and incubated with corresponding IRDye800 conjugated secondary antibodies (Licor) at 1:5000 for one hour at room temperature. Proteins were visualized using Odyssey infrared imaging system (Licor).
To generate rabbit antibody against thymidine kinase (TK or ORF21), the N-terminal region (amino acid 1–300) was cloned into pGEX-4T-1. Expression and purification of GST fusion proteins were carried out as previously described [47 (link),48 (link)] Purified proteins were used to immunize rabbits and whole serum was used to probe TK expression from γHV68-infected cells, along with pre-immune serum as a control. Anti-RTA antibody was described previously [49 (link)]. Rabbit sera against vGAT (ORF75c) and mLANA (ORF73) were described previously [50 (link),51 (link)]. The antibodies were affinity purified using GST fusion antigens.
To generate rabbit antibody against thymidine kinase (TK or ORF21), the N-terminal region (amino acid 1–300) was cloned into pGEX-4T-1. Expression and purification of GST fusion proteins were carried out as previously described [47 (link),48 (link)] Purified proteins were used to immunize rabbits and whole serum was used to probe TK expression from γHV68-infected cells, along with pre-immune serum as a control. Anti-RTA antibody was described previously [49 (link)]. Rabbit sera against vGAT (ORF75c) and mLANA (ORF73) were described previously [50 (link),51 (link)]. The antibodies were affinity purified using GST fusion antigens.
3
Lung Protein Expression Analysis
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Left lung lobe extracts were prepared in RIPA Buffer with Protease Inhibitor (Sigma‐Aldrich), electrophoresed by SDS‐PAGE with Tris Carboxy Ethyl Phosphene (TCEP; Thermo Fisher Scientific), electrotransferred to a Fluorescence PVDF membrane (MilliporeSigma), blocked for 60 min (Blocking Buffer; LI‐COR Biosciences) and probed with the following antibodies: IL‐33 (mouse mAb, 1:200 dilution, Nessy‐1 ALX‐804‐840/1; Enzo Life Sciences, Inc.), ST2 (goat pAb, 1:1000, Human ST2/IL‐33R AF523; R&D Systems), MYD88 (goat pAb, 1:1000, Mouse/Rat MyD88 AF3109; R&D Systems) and α‐tubulin (rabbit Ab, 1:2000 dilution; Cell Signaling Technology) overnight at 4°C. Signals were detected with IRDye800‐conjugated secondary antibodies (LI‐COR Biosciences) and visualized using the LI‐COR Odyssey imaging system (LI‐COR Biosciences). Signals were quantified using Image J and normalized to α‐tubulin.
4
Exogenous and Endogenous Protein Co-IP
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For coimmunoprecipitation (Co-IP) using exogenous protein, HEK293T cells were transfected with indicated expression plasmids for 48 h. For Co-IP using endogenous proteins, cells were directly harvested. Whole-cell lysates (WCLs) were prepared with NP-40 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 5 and mM EDTA) supplemented with 20 mM β-glycerophosphate and 1 mM sodium orthovanadate. WCLs were then sonicated, centrifuged, and precleared with Sepharose 4B for 1 h. Precleared samples were incubated with indicated antibodies overnight and with protein A/G agarose for 1 h at 4°C, or with antibody/glutathione-conjugated agarose for 4 h at 4°C. Agarose beads were washed extensively, and samples were eluted with SDS-PAGE loading buffer at 95°C for 10 min. The precipitated proteins were analyzed by SDS-PAGE and immunoblotting.
Immunoblotting was performed with the corresponding primary antibodies (1:1,000 dilution), as indicated in each figure, and IRDye800-conjugated secondary antibodies (1:10,000 dilution; LI-COR), unless specified otherwise. Proteins were visualized with an Odyssey infrared imaging system (LI-COR).
Immunoblotting was performed with the corresponding primary antibodies (1:1,000 dilution), as indicated in each figure, and IRDye800-conjugated secondary antibodies (1:10,000 dilution; LI-COR), unless specified otherwise. Proteins were visualized with an Odyssey infrared imaging system (LI-COR).
5
Immunoblotting Analysis of Mitochondrial Proteins
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Protein lysates for immunoblotting were prepared in RIPA buffer (Beyotime, Zhejiang, China) containing protease inhibitor cocktail tablets (Roche Applied Science, Mannheim, Germany), and the protein concentrations were quantified using the BCA protein assay kit (Pierce, Rockford, IL, USA). Western blot analysis was performed using standard techniques. The primary antibodies used were MCU (1:200; Santa Cruz, CA, USA), Bcl-2, Bax, Cytochrome (1:1000; Cell Signaling, Danvers, MA, USA) and GAPDH (1:5000; Beyotime, Zhejiang, China). The appropriate IRDye 800-conjugated secondary antibodies were used (1:10,000; LI-COR Biosciences, Lincoln, NE, USA) to visualize the blotting. Images were recorded using the Odyssey infrared imaging system and analyzed with Odyssey Application Software v2 (LI-COR Biosciences).
6
Comprehensive Western Blot Antibody Panel
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The following antibodies were used for Western blot staining procedures: αFMDV VP1 (rabbit polyclonal Abmade at PIADC), αEMCV capsid (gift from Ann Palmenberg), αLpro (gift from Ewald Beck and Tim Skern), αMAVS (Enzo life science ALX-210-929), αTBK1 (Cell signaling 3504), αIRF3 (Santa cruz sc-9082), αNF-κB-p65/RelA (Santa Cruz Biotechnology SC-8008), αeIF4G (Bethyl laboratories A300-502A), αG3BP1 (BD biosciences clone 23/G3BP), αPARP (Roche Diagnostics #11835238001), αFLAG (Sigma M2), αGFP (Invitrogen OSE00003G), αHis (GE Healthcare, 27-4710-01), αMyc (Clone 4A6, Millipore), αHA (Abcam ab130275) and αtubulin (Sigma DM1A). Respective IRdye680 or IRdye800 conjugated secondary antibodies (LiCOR) or HRP-conjugated secondary antibodies were used for detection.
7
Quantitative Western Blot Analysis
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For comparative protein quantification, cells were lysed with preheated SDS sample buffer, incubated for 15 min at 65 ºC, sonicated and centrifuged at 13,000 rpm for 15 min. A quantitative analysis of a Coomassie-stained SDS–polyacrylamide gel was used to adjust the total protein concentration of the lysates. Identical amounts of total cellular proteins were separated by SDS–polyacrylamide gel electrophoresis (Laemmli 1970 ) and transferred electrophoretically onto nitrocellulose membranes. Membranes were blocked with Tris-buffered saline-Tween (TBST: 0.02 M Tris, 0.150 M NaCl, 0.05% Tween 20) containing 5% non-fat dried milk or 3% BSA, and incubated overnight at 4 ºC with specific primary antibodies diluted in Tris-buffered saline with 0.05% Tween 20 (TBST) or in 2.5% non-fat milk powder in TBST. After washing 3 × 5 min with TBST, membranes were incubated either with IRDye-680- or IRDye-800-conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany) or with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and ECL Western Blotting Substrate (ThermoFisher/Pierce). Blots were analysed using a ChemiDoc MP Imaging System (Bio-Rad, Feldkirchen, Germany). Densitometric analysis was carried out using the Image Lab software (6.1.0; Bio-Rad) using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control.
8
Protein Extraction and Western Blot Analysis
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Protein extraction was performed using the M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Grand Island, NY, United States) and protein concentration was determined with the Bradford reagent (Sigma–Aldrich). Forty to 50 μg of protein extract for each sample were diluted in loading buffer, denatured by boiling at 95°C for 5 min. In the case of proteins extracted from MVs, an equal volume of the extracts was subjected to electrophoresis. Polyacrylamide-SDS gel at 10% or 4–15% were used. The proteins were then transferred onto nitrocellulose membranes (Hybond ECL, Amersham) through a semi-dry transfer apparatus for 60 min at 0.8 mA/cm2. After transfer, membranes were blocked with Odyssey blocking buffer (LI-COR Biotechnology GmbH, Bad Homburg, Germany) diluted 1:1 with phosphate-buffer, and then incubated with primary antibodies overnight, at 4°C. Immunodetection was performed using specific fluorescent IRDye©680 or IRDye©800-conjugated secondary antibodies (LI-COR). Detection of specific bands was carried out using the Licor Odyssey© infrared Imaging System (LI-COR). Band intensity was analyzed using the image processing software “ImageJ” developed by NIH and in public domain.
9
Co-Immunoprecipitation of Endogenous and Exogenous Proteins
10
Immunoprecipitation and Immunoblotting Protocol
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Commercial antibodies used in this study include mouse anti-HA monoclonal antibody and agarose (Sigma), mouse anti- β-Actin monoclonal antibody (Abcam), rabbit anti-COX-2 polyclonal antibody (Abcam), rabbit anti-RCAN1 polyclonal antibody (Sigma), mouse anti-SERCA2 (IID8) monoclonal antibody (Santa cruz), mouse anti-calcineurin Aα (Santa cruz). Thymidine kinase (TK) anti-serum was generated by immunizing rabbit with GST fusion protein containing N-terminal (aa1-330) of TK. RTA antibody were kindly provided by Dr. Yoshihiro Izumiya (UC-Davis). Immunoprecipitation and immunoblotting were carried out as described previously [37 (link)]. Briefly, cells were harvested and lysed with NP40 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 5 mM EDTA) supplemented with a protease inhibitor cocktail (Roche). Centrifuged cell lysates were pre-cleared with Sepharose 4B beads and incubated with HA-agarose at 4°C for 4 h. The agarose beads were washed three times with lysis buffer and precipitated proteins were released by boiling with 1×SDS sample buffer at 95°C for 5 min. Immunoblotting analysis was performed with the indicated primary antibodies and proteins were visualized with IRDye800 conjugated secondary antibodies (Licor) using an Odyssey infrared imaging system (Licor).
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