For immunoblotting (10 μg), proteins were electrotransferred onto nitrocellulose membranes using the iBlot™ Dry Blotting System (Life Technologies) and membranes blocked with 5% (w/v) skim milk powder in Tris-buffered saline with 0.05% (v/v) Tween-20 (TTBS) for 1 h. Membranes were probed with primary antibodies [mouse anti-ATP5B (Santa Cruz Biotechnology; 1:200), mouse anti-GPD2 (Santa Cruz Biotechnology; 1:200), rabbit anti-ARF4 (Abcam; 1:1000), rabbit anti-SDC2/HSPG2 (OriGene; 1:500)] for 10 h in TTBS (50 mM Tris, pH 7, 150 mM NaCl, 0.05% (v/v Tween 20) at 4°C, followed by incubation with either IRDye 800 goat anti-mouse IgG or IRDye 700 goat anti-rabbit IgG (1:15000, LI-COR Biosciences). Fluorescent signals were detected using the Odyssey Infrared Imaging System, (v3.0, LI-COR Biosciences, Nebraska USA).
Irdye 700 goat anti rabbit igg
Manufactured by LI COR
Sourced in United States
IRDye 700 goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. It is labeled with the IRDye 700 fluorescent dye, which can be detected using near-infrared imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800 goat anti mouse igg, by LI COR (11 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Odyssey clx infrared imaging system, by LI COR (3 mentions)
Mouse anti gapdh, by Thermo Fisher Scientific (3 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Image studio software, by LI COR (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ybx1, by Abcam (2 mentions)
Tsg101, by BD (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Mouse anti atp5b, by Santa Cruz Biotechnology (1 mentions)
Top2a, by Cell Signaling Technology (1 mentions)
Odyssey clx system, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Mouse anti vimentin, by Merck Group (1 mentions)
Rabbit anti snail1, by Abcam (1 mentions)
11 protocols using irdye 700 goat anti rabbit igg
1
Quantitative Protein Analysis by Immunoblotting
2
Quantitative Immunoblotting of Tumor Proteins
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SDS-PAGE and Western blot was performed on tumor cell lysates by the methods described previously21 (link). For immunoblotting, cell lysates (n = 4) (10 μg) resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes were probed with primary antibodies [mouse anti-human (1:1000)], TOP2A (Cell Signaling Technology, Danvers, MA, USA), PYCR2 and PPL (Santa Cruz Biotechnology, Dallas, USA) for 1 h in TTBS [50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20] followed by incubation with corresponding secondary antibodies; IRDye 800 goat anti-mouse IgG or IRDye 700 goat anti-rabbit IgG (1:15000, LI-COR Biosciences), for 1 h at room temperature in TTBS. Immunoblots were imaged using the CLx Odyssey Infrared Imaging System, (v3.0, LI-COR Biosciences, Nebraska USA). Loading controls were obtained by staining the membrane with Deep Purple Total Protein stain as previously described15 (link)16 (link). Semi-quantitative densitometric analysis was performed on all blots (3 biological replicates) to determine the level of protein expression using ImageStudio v5, with mean pixel intensity of the protein of interest normalised to the background.
3
Subcellular Fractionation and Western Blot Analysis
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PDAC cells were serum-starved for 48 h and treated as mentioned in the text. Sub-cellular fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific) as per the manufacturer’s instructions. Where indicated, the nuclear insoluble fraction was prepared by lysing the nuclear pellet in radioimmunoprecipitation assay buffer buffer and sonicating for five rounds of 30 s. Protein concentration was quantitated using the bicinchoninic protein assay kit (Pierce). Equal amounts of protein were loaded in each lane and separated on a 4–12% Bis-Tris NuPAGE gel (Invitrogen), then transferred onto a PVDF membrane. Membranes were probed with primary antibodies and infrared secondary antibodies: IRDye 700 goat anti-rabbit IgG or IRDye 800 goat anti-mouse IgG (LI-COR Biosciences). For protein band quantitation, infrared signals were detected using the Odyssey CLx infrared imaging system and bands quantified using Image Studio software (LICOR Biosciences).
4
Subcellular Fractionation and Western Blot
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PDAC cells were serum starved for 48 h and treated as described in the text. In accordance with the manufacturer’s instructions, subcellular fractions were prepared using Thermo Fisher Scientific’s NE-PER Nuclear and Cytoplasmic Extraction reagents. Bicinchoninic protein assay kits (Pierce) were used to measure protein concentrations. We loaded equal amounts of protein in each lane, separated the proteins on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and transferred the proteins to PVDF membranes. The following primary antibodies and infrared secondary antibodies were used to probe the membranes: IRDye 700 goat anti-rabbit IgG and IRDye 800 goat anti-mouse IgG (LI-COR Biosciences). LI-COR Biosciences’ Image Studio software (LI-COR) was used to quantify protein bands using infrared signals detected by the Odyssey CLx infrared imaging system.
5
Western Blot Protein Quantification Protocol
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Cells were lysed in RIPA buffer (Sigma) with phosphatase and protease inhibitors (EMD Millipore). Protein content was measured by bicinchoninic acid (BCA) assay and used to normalize samples to the lowest concentration. Lysates were heated to 95°C and run on a 4-12% Bis-Tris gel (Life Technologies) and transferred to a nitrocellulose membrane. Membranes were blocked in Odyssey blocking buffer (Li-Cor) and incubated with primary antibody at 4°C overnight (Table S2 ). All membrane wash steps were performed using Tris-buffered saline with 0.1% Tween-20. Membranes were incubated with secondary antibody, IRDye 800 goat anti-mouse IgG and IRDye 700 goat anti-rabbit IgG (Li-Cor) at a 1:10,000 dilution in blocking buffer for 1 h at room temperature. Blot fluorescence was visualized using an Odyssey CLx system (Li-Cor) and quantified with the built-in gel analyzer tool in ImageJ.
6
Protein Quantification and Immunoblotting Protocols
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Protein quantification was performed using 1D-SDS-PAGE / SYPRO® Ruby protein staining densitometry, as previously described [23 (link)]. For immunoblotting (10 μg), membranes were probed with primary antibodies [mouse anti-E-cadherin (BD Transduction Laboratories; 1:1000), mouse anti-H-Ras (Santa Cruz Biotechnology; 1:1000), rabbit anti-YBX1 (Abcam; 1:1000), mouse anti-Vimentin (Merck Millipore; 1:1000), mouse anti-β-actin (Cell Signalling Technologies; 1:2000), mouse anti-Claudin1 (Santa Cruz Biotechnology; 1:1000), rabbit anti-Snail1 (Abcam; 1:1000), rabbit anti-Twist (Santa Cruz Biotechnology; 1:1000), or mouse anti-GAPDH (Life technologies; 1:12, 000) for 1 hr at RT in TTBS (50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20) followed by incubation with either IRDye 800 goat anti-mouse IgG or IRDye 700 goat anti-rabbit IgG (1:15000, LI-COR Biosciences) for 1 hr at RT in TTBS. Immunoblots were imaged using the Odyssey Infrared Imaging System, (v3.0, LI-COR Biosciences, Nebraska USA).
7
Protein Quantification and Immunoblotting
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Protein samples were quantified by 1D SDS-PAGE/SYPRO Ruby protein staining-based densitometry18 (link). Western blotting was performed on protein samples (10–20 µg) as previously described18 (link). Rabbit antibodies raised against GAPDH (Cell Signalling), β-tubulin (Cell Signalling), KRASG12V mutant specific (Cell Signalling), RAB7 (Abcam) and GFP (Abcam) were used. Mouse antibodies against MKLP1 (Santa Cruz), RACGAP1 (Santa Cruz), ALIX (BD Biosciences), TSG101 (BD Biosciences), CD63 (Santa Cruz), CD81 (Santa Cruz), RAB2A (Thermo Fisher), FLOT1 (BD Biosciences), α-actinin (Abcam), CD9 (Santa Cruz) and HSP90 (BD Biosciences) were used. Secondary antibodies used were IRDye 800 goat anti-mouse IgG or IRDye 700 goat anti-rabbit IgG (1:15000, LI-COR Biosciences).
8
Protein Quantification and Immunoblotting Protocol
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Protein content was estimated by 1D-SDS-PAGE/SYPRO Ruby protein staining-based densitometry, as previously described [22 (link)]. For immunoblotting (10 μg), membranes were probed with primary antibodies [mouse anti-Rac1 (Abcam, 1:1000), mouse anti-Alix mouse (Cell Signalling, 1:1000), mouse anti-TSG101 (Cell Signalling, 1:1000), and mouse anti-GAPDH (Ambion, 1:12,000)] for 1 h in TTBS (50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20) followed by incubation with corresponding secondary antibodies; IRDye 800 goat anti-mouse IgG or IRDye 700 goat anti-rabbit IgG (1:15000, LI-COR Biosciences), for 1 h at RT in TTBS. Immunoblots were imaged using the Odyssey Infrared Imaging System, (v3.0, LI-COR Biosciences, Nebraska USA).
9
Quantitative Protein Analysis and Immunoblotting
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Protein quantification (microBCA™ Protein Assay Kit (23235, Thermo Fisher Scientific)) and Western blotting (iBlot 2 Dry Blotting System, Thermo Fisher Scientific) were performed as per manufacturer's instructions. Dot blot analysis was performed using 96‐well Bio‐Dot (Bio‐Rad Laboratories) as per manufacturer's instructions with proteins were lysed in 50 mM HEPES (1% SDS). Rabbit antibodies raised against, MET (Santa Cruz Biotechnology), CD63 (Santa Cruz), ANXA1 (Abcam), GAPDH (Cell Signalling), and GFP (Abcam) were used. Mouse antibodies ALIX (BD Biosciences), TSG101 (BD Biosciences) were used. Secondary antibodies used were IRDye 800 goat anti‐mouse IgG or IRDye 700 goat anti‐rabbit IgG (1:15000, LI‐COR Biosciences).
10
Quantification and Immunoblotting of Proteins
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Protein quantification was performed using 1D-SDS-PAGE/SYPRO Ruby protein staining densitometry, as previously described33 (link). For immunoblotting (10 μg), membranes were probed with primary antibodies [mouse anti-CDH1 (BD Biosciences; 1:1000), rabbit anti-MMP1 (Santa Cruz Biotechnology; 1:1000), mouse anti-VIM (Cell signalling; 1:1000), rabbit anti-YBX1 (Abcam; 1:1000), mouse anti –FN1 (Sigma, 1:1000), rabbit anti-LAMA5 (Santa Cruz Biotechnology; 1:1000), rabbit anti-COL12A1 (Santa Cruz Biotechnology; 1:1000), rabbit anti-p44/42 MAPK (ERK 1/2) (Cell signalling; 1:1000), rabbit anti-phospho-p44/42 MAPK (p-ERK 1/2) (Cell Signalling; 1:1000), or mouse anti-GAPDH (Life technologies; 1:12,000)] for 1 hr at RT in TTBS (50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20 in PBS) followed by incubation with the corresponding secondary antibodies (IRDye 800 goat anti-mouse IgG or IRDye 700 goat anti-rabbit IgG (1:15,000, LI-COR Biosciences) for 1 hr at RT in TTBS. Immunoblots were imaged and visualised using the Odyssey Infrared Imaging System, (v3.0, LI-COR Biosciences, Nebraska USA).
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