Cells were lysed in Hypotonic Lysis Buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, and cOmplete mini protease inhibitor (Sigma-Aldrich) for 20 min on ice. The lysates were mixed 1:1 with 2 × Laemmli buffer containing 1:20-diluted 2-mercaptoethanol, boiled for 10 min, and centrifuged at 16,000 × g for 5 min at 4°C. Samples were run on 4–20% SDS-PAGE and transferred to nitrocellulose membranes. Membrane blocking, as well as antibody binding were in TBS Odyssey Blocking Buffer (Li-Cor, Lincoln, NE). Primary antibodies used were rabbit anti-CypA (1:10,000 dilution; Enzo Life Sciences, Farmingdale, NY, catalogue #BML-SA296) and mouse anti-β-actin (1:1,000 dilution; Abcam, Cambridge, UK, catalogue #ab3280). Goat anti-mouse-680 (Li-Cor, catalogue #925–68070) and goat anti-rabbit-800 (Li-Cor, catalogue #925–32211) as secondary antibodies were used at 1:10,000 dilutions. Blots were scanned on the Li-Cor Odyssey CLx.
Goat anti mouse 680
Manufactured by LI COR
The Goat anti-mouse 680 is a secondary antibody that binds to mouse primary antibodies. It is labeled with the fluorescent dye IRDye 680RD, which can be detected using near-infrared imaging platforms.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit 800, by LI COR (14 mentions)
Donkey anti rabbit 800, by LI COR (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Complete mini protease inhibitor, by Merck Group (2 mentions)
Odyssey blocking buffer tbs, by LI COR (2 mentions)
Mouse anti β actin, by Abcam (2 mentions)
Rabbit anti cypa, by Enzo Life Sciences (2 mentions)
Odyssey clx, by LI COR (2 mentions)
Monoclonal mouse anti puromycin clone 12d10, by Merck Group (2 mentions)
Rabbit anti gapdh abs16, by Merck Group (2 mentions)
Recombinant trail ligand 168 amino acid tnf homologous extracellular domain, by Thermo Fisher Scientific (2 mentions)
Ab18251, by Abcam (2 mentions)
Tigar, by Abcam (2 mentions)
Ab228633, by Cell Signaling Technology (2 mentions)
Anti actin, by Cell Signaling Technology (2 mentions)
Odyssey imager, by LI COR (2 mentions)
Trans blot turbo, by Bio-Rad (2 mentions)
Blocking buffer, by LI COR (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
23 protocols using goat anti mouse 680
1
Protein Extraction and Western Blotting
2
SARS-CoV-2 Infection and Host Response in Calu-3 Cells
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Calu-3 cells were seeded at a density of 3 x 105 cells/well in 12-well plates. Cells were infected with SARS-CoV-2 at an MOI of 1. Control cells were sham infected. Twelve hours post incubation, cells were transfected or treated with poly(I:C) or IFNβ, respectively for 6 h. Cell lysates were harvested for immunoblots and analyzed on reducing gels as mentioned previously (Banerjee et al., 2020b (link), 2021 (link)). Briefly, samples were denatured in a reducing sample buffer and analyzed on a reducing gel. Proteins were blotted from the gel onto polyvinylidene difluoride (PVDF) membranes (Immobilon, EMD Millipore) and detected using primary and secondary antibodies. Primary antibodies used were: 1:1000 mouse anti-SARS/SARS-CoV-2 N (ThermoFisher Scientific; Catalog number: MA5-29981; RRID: AB_2785780 ), 1:1000 rabbit anti-beta-actin (Abcam; Catalog number: ab8227; RRID: AB_2305186 ), and 2 μg/mL of mouse anti-ACE2 (R&D Systems; Catalog: MAB933; RRID: AB_2223153 ). Secondary antibodies used were: 1:5000 donkey anti-rabbit 800 (LI-COR Biosciences; Catalogue number: 926-32213; RRID: 621848) and 1:5000 goat anti-mouse 680 (LI-COR Biosciences; Catalogue number: 925-68070; RRID: AB_2651128 ). Blots were observed and imaged using Image Studio (LI-COR Biosciences) on the Odyssey CLx imaging system (LI-COR Biosciences).
3
Evaluating TRAIL-Induced Apoptosis
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Unless otherwise noted, general reagents were obtained from Sigma (St. Louis, MO), cell culture reagents and electrophoresis/western blot supplies were from Invitrogen (Carlsbad, CA) and cell culture plasticware from Corning (Corning, NY). Monoclonal mouse anti-puromycin clone 12D10 [1 (link)] and rabbit anti-GAPDH (ABS16) were obtained from EMD-Millipore (Billerica, MA). Monoclonal mouse anti-cFLIP clone 7F10 was obtained from Enzo (Farmingdale, NY). Secondary antibodies, Goat anti-rabbit (800) and Goat anti-mouse (680) and blocking buffer were from LiCor (Lincoln, NE). Sources of other reagents: 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT; NSC 601519) from the NCI Drug Synthesis and Chemistry Branch; recombinant TRAIL ligand (168 amino acid TNF homologous extracellular domain—Peprotech). Caspase 8 assay kit (CaspaseGlo 8) was obtained from Promega (Madison, WI) and was used according the manufacturer’s directions.
4
Western Blot Analysis of pERK/tERK
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Cell lysate protein was quantified with a modified BCA protein assay using manufacturers protocol (Bio-Rad). Samples (20–30 μg) were ran on precast gels (10% Bis–Tris, Bolt brand, from ThermoFisher). Gels were wet-transferred to nitrocellulose membranes at 30 V for at least 60 min at 4 °C. Blots were blocked with 5% non-fat dry milk in TBS for 30 min, washed 3 × for 5 min with TBS + 0.1% Tween-20 (TBST), and incubated with primary antibody overnight at 4 °C. Primary antibody: pERK and tERK (Cell Signaling) at 1:1000 dilution in 5% BSA in TBST. Blots were then washed 3 × with TBST then incubated with secondary antibodies for 90 min at room temperature. Secondary antibodies: Goat anti-Rabbit 800 W and Goat anti-Mouse 680 diluted at 1:10,000 and 1:20,000 (Licor), respectively, in 5% non-fat milk in TBST. Blots were washed 3X with TBST and 1 × with TBS, then imaged on a Licor Odyssey Fc or Azure Sapphire. Bands were analyzed using Image-J and reported as pERK/tERK normalized to the standard, or simply as pERK/tERK.
5
Protein Extraction and Western Blotting
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Cells were lysed in Hypotonic Lysis Buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, and cOmplete mini protease inhibitor (Sigma-Aldrich) for 20 min on ice. The lysates were mixed 1:1 with 2 × Laemmli buffer containing 1:20-diluted 2-mercaptoethanol, boiled for 10 min, and centrifuged at 16,000 × g for 5 min at 4°C. Samples were run on 4–20% SDS-PAGE and transferred to nitrocellulose membranes. Membrane blocking, as well as antibody binding were in TBS Odyssey Blocking Buffer (Li-Cor, Lincoln, NE). Primary antibodies used were rabbit anti-CypA (1:10,000 dilution; Enzo Life Sciences, Farmingdale, NY, catalogue #BML-SA296) and mouse anti-β-actin (1:1,000 dilution; Abcam, Cambridge, UK, catalogue #ab3280). Goat anti-mouse-680 (Li-Cor, catalogue #925–68070) and goat anti-rabbit-800 (Li-Cor, catalogue #925–32211) as secondary antibodies were used at 1:10,000 dilutions. Blots were scanned on the Li-Cor Odyssey CLx.
6
Myc-Borealin Expression Analysis in HeLa Cells
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To study the expression levels of each of the Myc-Borealin constructs, HeLa Kyoto cells were transfected in 12-well dishes as described above and solubilized after 36 h in 1× Laemmli buffer, boiled for 5 min, and analyzed by SDS-PAGE followed by Western blotting. The antibodies used for the immunoblot were rabbit anti-tubulin (1:10,000; ab18251; Abcam), mouse anti-myc (1:1,000; CSB-MA000041Mom, Cusabio), and mouse anti-Borealin (1:1,000; M147-3; MB). Secondary antibodies used were goat anti-mouse 680 and donkey anti-rabbit 800 (LI-COR) at 1:2,000 dilution. Immunoblots were imaged using the Odyssey CLx system.
7
Protein Extraction and Western Blotting
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Approximately 4 million cells were harvested with RIPA lysis buffer with phosphatase inhibitor (Thermo Scientific, no. A32957) and HALT protease inhibitor (Thermo Scientific, no. 78425). Proteins of interest were probed with the following antibodies in TBS-T buffer with 5% BSA. Primary antibodies: G6PD (abcam, no. ab993), TIGAR (abcam, no. ab37910), Beta-actin (Cell Signaling, no. 3700 S); and secondary antibodies: Goat-anti-rabbit 800 (LI-COR, no. 925-32211), Goat-anti-mouse 680 (LI-COR, no. 925-68070).
8
Western Blot Analysis of Adpgk and Actin
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Cell lysates containing ~30 μg protein were loaded on a 15-well 4 to 12% NuPage gel and electrophoresed for 45 min at 150V. Proteins were transferred to nitrocellulose using the Transblot Turbo (BioRad). Blots were rinsed with water and dried, reconstituted with TBS, and blocked with Odyssey TBS blocking buffer for 1 h. Antibodies were added at 1:1000 dilution (anti-Adpgk, Abcam, ab228633) or 1:5000 dilution (anti-actin, Cell Signaling Technology, 3700) in TBS blocking buffer, 0.2% Tween-20 and rocked overnight at 4 °C. Blots were then rinsed four times with TBS, 0.1% TBST. Secondary antibodies were added at 1:15,000 dilution (LI-COR, goat anti-mouse 680, goat anti-rabbit 800) for 1 h at room temperature. Blots were rinsed three times with TBS, 0.1% TBST, then once with TBS. Images were obtained using an Odyssey imager (LI-COR).
9
Western Blot Analysis of Smad Signaling
10
Fibronectin and Actin Detection in Cell Lysates
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