Whole-cell extracts were prepared from cultures at different time points by boiling bacterial suspensions in SDS-PAGE lysis buffer (35 ) at a dilution of 1 ml per OD600 unit. Proteins were separated by SDS-PAGE alongside molecular weight markers (All Blue; Bio-Rad) and transferred to nitrocellulose membranes (Hybond-C Extra; Amersham) for Western blotting. PilE was detected using SM1 (1:10,000) (36 (link)) and IRDye 800CW–goat anti-mouse IgG (1:10,000; Li-Cor). A GroEL loading control was detected using anti-GroEL antibody (1:8,000; gift from Jörgen Johansson) and IRDye 680–goat anti-rabbit IgG (1:10,000; Li-Cor). RecA was detected using an anti-RecA antibody (1:6,000; Abcam) and IRDye 680–goat anti-rabbit IgG (1:10,000; Li-Cor). Bands were visualized and quantified using the Odyssey Sa infrared imaging system. Experiments were carried out in triplicate using strains from independent transformations. PilE band intensities were normalized to the respective GroEL band intensities and expressed as the ratio to the normalized PilE value of the first lane.
Irdye 680 goat anti rabbit igg
Manufactured by LI COR
Sourced in United States
IRDye 680 goat anti-rabbit IgG is a secondary antibody conjugated with the near-infrared dye IRDye 680. It is designed for detection of rabbit primary antibodies in western blotting, immunohistochemistry, and other immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800 goat anti mouse igg, by LI COR (17 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (16 mentions)
Odyssey infrared imaging system, by LI COR (14 mentions)
Odyssey blocking buffer, by LI COR (11 mentions)
Odyssey infrared imager, by LI COR (4 mentions)
Odyssey imaging system, by LI COR (3 mentions)
Odyssey clx imaging system, by LI COR (3 mentions)
Rabbit anti tsg101, by Thermo Fisher Scientific (2 mentions)
Rabbit anti cd63, by Santa Cruz Biotechnology (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Mouse anti β actin, by Novus Biologicals (2 mentions)
Rabbit polyclonal anti kar2 y 115, by Santa Cruz Biotechnology (2 mentions)
Mouse monoclonal anti myc, by Abcam (2 mentions)
Hrp conjugated mouse monoclonal anti flag m2, by Merck Group (2 mentions)
Mouse monoclonal anti mbp, by New England Biolabs (2 mentions)
Mouse anti gapdh, by Merck Group (2 mentions)
Nitrocellulose membrane, by LI COR (2 mentions)
Goat anti mouse igg dylight 488, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg dylight 550, by Thermo Fisher Scientific (2 mentions)
Sybr green taqman pcr kit, by Thermo Fisher Scientific (2 mentions)
41 protocols using irdye 680 goat anti rabbit igg
1
Quantitative Analysis of Pilus Protein Expression
2
Quantification of M6A Regulators in Heart Tissue
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The protein component of the heart tissue was lysed with RIPA lysis buffer (Alfa Aesar). After quantifying by BCA protein kit (Thermo Scientific), equal amounts of protein were loaded on a 10% or 12% sodium dodecyl sulfate-polyacrylamide gel and separated by electrophoresis. Then, the protein was transferred to a nitrocellulose membrane. After blocking with Odyssey blocking buffer (LI-COR Biosciences), membranes were incubated with rabbit anti-Mettl3 (1:1,000, Proteintech), rabbit anti-Mettl14 (1:1,000, ABclonal), rabbit anti-WTAP (1:1,000, Cell Signaling Technology), rabbit anti-ALKBH5 (1:1,000, NOVUS Biologicals), rabbit anti-FTO (1:1,000, NOVUS Biologicals), and mouse anti-GAPDH (1:5,000, MilliporeSigma) overnight on a rocker at 4°C. Membranes were then incubated for 1 h at room temperature with IRDye 680 goat anti-rabbit IgG and IRDye 800 Goat anti-mouse IgG (1:10,000, LI-COR Biosciences). The probed blots were scanned using an Odyssey infrared imager (LI-COR Biosciences).
3
Protein Expression Analysis by Western Blot
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Total cell lysates (25 μg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore) and immunoblotted with the indicated antibodies, followed by IRDye800CW goat anti-mouse IgG or IRDye680 goat anti-rabbit IgG (LI-COR Biosciences). Blots were visualized on an Odyssey Infrared Imaging System (LI-COR Biosciences).
4
Antibody Identification for Signaling Pathway
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Anti-MLK3 (A-20) (for detection of murine MLK3), anti-FRA-1 (R-20), anti-JNK1/3 (C-17), anti-ERK1 (K-23), anti-P38 (C-20), anti-actin (C-2), anti-p-c-JUN (S63)(KM-1) and anti-c-JUN (H-79) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-MLK3 (C-terminal) (for detecting human MLK3) was from Epitomics (Cambridge, MA, USA). Anti-p-MLK3, anti-p-ERK-1/2 (T202/Y204)(E-10), anti-p-JNK1/2 (T183/Y185)(81E11) and anti-p-P38 (T180/Y182) (#9216) were obtained from Cell Signaling (Danvers, MA, USA). Anti-MMP-1 (#36665 R) was purchased from R&D systems (Minneapolis, MN, USA), anti-p-paxillin S178 (#A300-100 A) was purchased from Bethyl Laboratory (Montgomery, TX, USA). IRDye 800CW goat anti-mouse IgG, IRDye 680 goat anti-rabbit IgG and IRDye 800CW donkey anti-goat IgG were from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit IgG conjugated with Alexa Fluor 488 and 546 was from Invitrogen and used for immunofluorescence staining.
5
Analyzing Apoptosis Signaling Pathways
6
Immunoblot Analysis of Hepatitis C Virus Proteins
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A standard immunoblot procedure was employed [22] . Protein samples transferred to PVDF membranes were probed with the following primary antibodies: anti-core (1∶2,000, Affinity BioReagents, MA1-080), 9E10 (kindly provided by Dr. Charles Rice and Dr. Tim Tellinghuisen), anti-NS3 (1∶1,000, Virogen, 217-A), anti-CKIIα (1∶2,500, Bethyl Laboratories, A300-197A), anti-CKIIα′ (1∶2,500, Bethyl Laboratories, A300-199A), and anti-GAPDH (1∶500,000, Ambion, AM4300) antibodies. Proteins were visualized with IRDye 800CW Goat anti-Mouse IgG or IRDye 680 Goat anti-Rabbit IgG, and images collected on an Odyssey infrared imaging system (LI-COR Biosciences).
7
Exosome Protein Analysis via Western Blotting
8
Western Blot Analysis of Notch1, FBXW7, and Caspase 3
9
Quantitative Protein Profiling of EVs
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Protein content was estimated by 1D-SDS-PAGE/SYPRO® Ruby protein staining densitometry, as previously described173 (link). For immunoblotting (10 μg), membranes were probed with primary antibodies [mouse anti-TSG101 (BD Transduction Laboratories; 1:500), mouse anti-Alix (Cell Signaling Technology; 1:1000), rabbit anti-CD70 (Abcam; 1:1000), mouse anti-HSPG1 (Abcam; 1:200), rabbit anti-MSLN (Cell Signaling Technology; 1:1000), rabbit anti-FAT1 (GeneTex; 1:2000), mouse anti-CD81 (Santa Cruz Biotechnology; 1:1000), rabbit anti-CALR (Abcam; 1:1000) for 3 hr at room temperature (RT) in 50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20 (TTBS) followed by incubation with either IRDye 800 goat anti-mouse or goat IgG or IRDye 680 goat anti-rabbit IgG (1:15000, LI-COR Biosciences) for 1 hr at RT in TTBS. Immunoblots were visualized using the Odyssey Infrared Imaging System and Image Studio™ Software (v3.0, LI-COR Biosciences, Nebraska USA).
10
Histone H1 Quantification Protocol
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Histone samples were exposed to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (14%), transferred to a PVDF membrane, blocked with Odyssey blocking buffer (LI-COR Biosciences) or 5% non-fat milk for 1 hr, and incubated with primary antibodies overnight at 4°C as well as with secondary antibodies conjugated to fluorescence (IRDye 680 goat anti-rabbit IgG or IRDye 800 goat anti-mouse IgG, Li-Cor) for 1 hr at room temperature. Bands were visualized in an Odyssey Infrared Imaging System (Li-Cor). Coomassie staining or histone H3/histone H4 immunoblotting were used as loading controls. H1 protein content was quantified from Coomassie staining of histone extracts using ImageJ software. H1 variants can be visualized in three consecutive bands (35–32 kDa, corresponding to H1.3 + H1.4 + H1.5, H1.2, and H1.0, respectively), as indicated in Figure 5—figure supplement 1A, C . H1X cannot be quantified from Coomassie staining. The relative intensity of each H1 band was corrected by H4 band (loading control) and expressed as a percentage of total H1 content.
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