Total cell extracts from freeze-clamped, pulverised tissues were obtained by incubation on ice for 10 min in cell lysis buffer (RIPA, Pierce) supplemented with proteinase inhibitors (1 × Complete (Roche), 1:500 PI cocktail (Sigma)) and 10 mM sodium butyrate (Sigma)). Lysates were sonicated (Bioruptor; 3 × 30 s, high setting) and spun at 13000 rpm, 4 °C for 30 s. Thirty microgram protein extracts were run on 4–20% Bis-Tris gradient gels (Invitrogen), transferred onto PVDF membranes (GE Healthcare) and stained with Ponceau S (Sigma). The blotted membranes were cut at ~40 KDa and blocked with 3% skimmed milk in TBS-T (TBS/0.02% Tween-20) for 1 h at room temperature. All antibody incubations were in 1% milk/TBS-T for 1 h at room temperature and washes between antibodies in TBS-T. The bottom half was incubated with rabbit anti-H3Ac (Millipore, 06-599, 1:10,000) and the top half with rabbit anti-LaminB1 (Abcam, ab16408, 1:1000). After 3 × 5 min washes, top and bottom half membranes were incubated with donkey anti-rabbit IR-Dye 800 (LI-COR, 926-32213), washed and scanned on a LI-COR Odyssey scanner. Fluorescence intensity signals were quantified with ImageQuant (GE Healthcare).
Donkey anti rabbit irdye 800
Manufactured by LI COR
Sourced in United States
The Donkey anti-rabbit IRDye 800 is a fluorescent-labeled secondary antibody used for the detection and quantification of rabbit primary antibodies in western blot, immunohistochemistry, and other immunoassay applications. The IRDye 800 fluorescent label allows for near-infrared detection and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti mouse irdye 680, by LI COR (6 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Donkey anti mouse irdye800, by LI COR (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Odyssey clx infrared scanner, by LI COR (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti misp, by Thermo Fisher Scientific (3 mentions)
Anti villin, by Santa Cruz Biotechnology (3 mentions)
Gtx125989, by GeneTex (3 mentions)
Gtx125923, by GeneTex (3 mentions)
Irdye 680 goat anti rat, by LI COR (3 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (2 mentions)
Mouse anti rsv n, by Abcam (2 mentions)
Irdye 680rd donkey anti mouse, by LI COR (2 mentions)
Mouse monoclonal anti gapdh 6c5, by Merck Group (2 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
24 protocols using donkey anti rabbit irdye 800
1
Quantitative Immunoblotting of Histone Acetylation
2
Quantification of D4 Receptor Expression in Hypertensive Rats
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We next compared the expression of D4 receptor in the RPT cells from WKY rats and SHRs. Total protein (50 µg) was separated by electrophoresis on 10% or 15% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes (Amersham Life Science, Arlington, Texas, USA). The blots were blocked overnight and then incubated with polyclonal rabbit anti-rat D4 receptor antibody (1:400; Bioss, Woburn, Massachusetts, USA) overnight at 4°C. The membranes were washed with phosphate buffer saline-tween and then incubated with infrared-labeled secondary antibody (donkey anti-rabbit IRDye 800, Li-Cor Biosciences, Lincoln, Nebraska, USA). The bound complex was determined using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, Nebraska, USA). The densities of bands were normalized against that of glyceraldehyde-3-phosphate dehydrogenase (n = 4) or NKA (n = 3).
3
Immunostaining and Western Blot Protocols
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For immunostaining, primary antibodies used were mouse anti-RSV N (Abcam, Cat. No. ab22501), mouse anti-RSV G (Abcam, Cat. No. ab94966), rabbit anti-camelid VHH (GenScript, Cat. No. A01860-200), goat anti-panRSV polyclonal (Millipore, Cat. No. AB1128), rat anti-CD63 (BD, Cat. No. 564221), rabbit anti-Rab5 (Affinity BioReagents, Cat. No. PA3-915), and rabbit anti-Rab7 (Abcam, Cat. No. 137029). All primary antibodies for immunostaining experiments were used at 1 µg/mL. Secondary antibodies used were donkey anti-human Alexa Fluor 647 (Jackson ImmunoResearch), donkey anti-goat Alexa Fluor 546, donkey anti-rabbit Alexa Fluor 488, and donkey anti-mouse Alexa Fluor 488 (all from Life Technologies). All secondary antibodies for immunostaining experiments were used at 4 µg/mL. For dSTORM experiments, secondary antibodies used were donkey anti-mouse Alexa Fluor 647 (Life Technologies) and goat anti-human CF568 (Biotium), and both were used at 1 µg/mL.
For western blot, the primary antibodies used were a rabbit monoclonal anti phosphorylated eIF2α (Cell Signaling Technologies, Cat. No. 9721) and goat anti-pan-eIF2α (R&D Systems, Cat. No. AF3997) diluted 1:1000 in 5% BSA in TBS with 0.1% Tween-20. The secondary antibodies were a donkey anti-mouse IRDye 680RD (LI-COR) and a donkey anti-rabbit IRDye 800 (LI-COR) and were diluted 1:5000 in 5% BSA in TBS with 0.1% Tween-20.
For western blot, the primary antibodies used were a rabbit monoclonal anti phosphorylated eIF2α (Cell Signaling Technologies, Cat. No. 9721) and goat anti-pan-eIF2α (R&D Systems, Cat. No. AF3997) diluted 1:1000 in 5% BSA in TBS with 0.1% Tween-20. The secondary antibodies were a donkey anti-mouse IRDye 680RD (LI-COR) and a donkey anti-rabbit IRDye 800 (LI-COR) and were diluted 1:5000 in 5% BSA in TBS with 0.1% Tween-20.
4
Immunostaining and Western Blot Protocols
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For immunostaining, the primary antibodies used were mouse anti-CD63 (Developmental Studies Hybridoma Bank, Cat. No. H5C6), mouse anti-clathrin LC (Biolegend, Cat. No. MMS-423P), mouse anti-caveolin (Abcam, Cat. No. ab17052), LAMP1 (Developmental Studies Hybridoma Bank, Cat. No. H4A3), mouse anti-EEA1 (BD Biosciences, Cat. No. 610456), and rabbit anti-NanoLuc (Promega). All primary antibodies for immunostaining experiments were used at 1 μg/mL. Secondary antibodies used were donkey anti-mouse Alexa Fluor 546 (Life Technologies) and donkey anti-rabbit Alexa Fluor 488 (Life Technologies). All secondary antibodies for immunostaining experiments were used at 8 μg/mL.
For western blot, primary antibodies used were rabbit anti-NanoLuc (Promega), and mouse anti-GAPDH (GeneTex, Cat. No. GT239), diluted to 0.74 μg/mL and 1 μg/mL, respectively, in Odyssey blocking buffer (LI-COR) with 0.1% Tween-20. The secondary antibodies were a donkey anti-mouse IRDye 680RD (LI-COR) and a donkey anti-rabbit IRDye 800 (LI-COR) and were diluted 1:3,000 in Odyssey blocking buffer with 0.1% Tween-20.
For western blot, primary antibodies used were rabbit anti-NanoLuc (Promega), and mouse anti-GAPDH (GeneTex, Cat. No. GT239), diluted to 0.74 μg/mL and 1 μg/mL, respectively, in Odyssey blocking buffer (LI-COR) with 0.1% Tween-20. The secondary antibodies were a donkey anti-mouse IRDye 680RD (LI-COR) and a donkey anti-rabbit IRDye 800 (LI-COR) and were diluted 1:3,000 in Odyssey blocking buffer with 0.1% Tween-20.
5
Western Blot Analysis of D1 Receptors
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After boiling the homogenates in sample buffer (35 mmol/L Tris-HCl, pH 6.8, 4% SDS, 9.3% dithiothreitol, 0.01% bromophenol blue, 30% glycerol) at 95°C for 5 min, 100 μg of protein were separated by SDS-PAGE (10% polyacrylamide), and then electroblotted onto nitrocellulose membranes (Bio-Rad). The blots were blocked overnight with 5% nonfat dry milk in phosphate buffered saline with Tween 20 (PBST) (0.05% Tween 20 in 10 mmol/l phosphate buffered (isotonic) saline) at 4°C with constant shaking, then incubated with polyclonal rabbit anti-rat D1 receptor antibodies (1:400 dilution; Millipore) overnight in the cold-room at 4°C. The membranes were then further incubated with infrared- labeled secondary antibodies (donkey anti-rabbit IRDye 800, Li-Cor Biosciences, Lincoln, NE) added to bind to the primary antibody at room temperature for 1 hr. The membranes were washed three times with PBST. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities.
6
Rab32 Expression Analysis in Infected THP-1 Cells
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PMA-differentiated THP-1 cells were infected as described above and lysed in SDS–polyacrylamide gel electrophoresis loading buffer 2.5 hours p.i. Western blot analysis was performed using the Odyssey Infrared Imaging System (LI-COR Biosciences). The following antibodies were used for Western blot analysis: rabbit polyclonal anti-Rab32 (GeneTex; 1:1000 dilution) and donkey anti-rabbit IR Dye 800 (LI-COR Biosciences; 1:10,000 dilution).
7
Western Blot Analysis of Cell Proteins
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Cultured cells were washed twice with 1 × PBS and then incubated for 1 min in urea buffer (8 M urea, 100 mM NaH2PO4 and 10 mM Tris pH 8), scraped, harvested and briefly sonicated. The proteins were subjected to SDS-polyacrylamide gel electrophoresis. The resolved proteins were blotted overnight onto nitrocellulose membranes, which then were blocked in 1 × PBS containing 5% non-fat milk for at least 1 h. The blots were incubated with the following primary anti-human antibodies: rabbit polyclonal anti-N-Myc (C-19; Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal anti-GAP43 (GAP-7B10; Sigma-Aldrich); rabbit polyclonal anti-CDK5 (Cell Signaling Technology, Danvers, MA, USA); mouse monoclonal anti-cyclin A (C-19; Santa Cruz Biotechnology); rabbit polyclonal anti-p27kip1 (C-19; Santa Cruz Biotechnology); goat polyclonal anti-ChAT (Millipore, Billerica, MA, USA); mouse monoclonal anti-GAPDH (6C5; Millipore) and mouse monoclonal anti-HSP 72/73 (Ab1-W27; Oncogene Science Inc., Cambridge, MA, USA). The membranes were then incubated for 45 min with the appropriate secondary antibody: donkey anti-rabbit IRdye800 (LI-COR Biosciences, Lincoln, NE, USA); donkey anti-mouse IRdye800 (LI-COR) or donkey anti-goat IRdye800 (LI-COR). The membranes were then analysed with a Licor Odyssey Infrared Image System in the 800 nm channel. Blot scan resolution was 150 d.p.i.
8
Western Blot Analysis of ER Stress Proteins
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Cell lysates were mixed with Laemmli buffer plus dithiothreitol (DTT), heated for 5 min at 95 °C, and loaded onto SDS-PAGE (sodium dodecyl sulfate—polyacrylamide gel electrophoresis) gels. The gels were run in Tris–Glycine SDS running buffer and then transferred onto nitrocellulose membranes. Blocking consisted of at 5% skim milk or 2.5% BSA, depending on the primary antibody used. Imaging was performed using the Li-Cor Odyssey fluorescence imaging system. Primary antibodies consisted of: monoclonal mouse anti-6xHis-Tag (Invitrogen, MA1-21315; 1:4000), polyclonal rabbit anti-ATF6 (Novus Biologicals, NBP1-75478; 1:1000), monoclonal rabbit anti-BiP (Cell Signaling Technology, C50B12; 1:1000), monoclonal rabbit anti-Calnexin (Cell Signaling Technology, C5C9; 1:1000), monoclonal rabbit anti-COG5 (Sigma-Aldrich, SAB4200440; 1:600), monoclonal mouse anti-GAPDH (Invitrogen, ZG003; 1:4000), monoclonal mouse anti-GFP (Cell Signaling Technology, 2955S; 1:1000), polyclonal rabbit anti-IRE1a (Cell Signaling Technology, 3294S; 1:1000), monoclonal rabbit anti-PDI (Cell Signaling Technology, 2446S; 1:1000), and monoclonal rabbit anti-PERK (Cell Signaling Technology, D11A8; 1:1000). Secondary antibodies consisted of: Donkey anti-Rabbit IRDye 680, Donkey anti-Rabbit IRDye 800, Donkey anti-Mouse IRDye 680, and Donkey anti-Mouse IRDye 800 (Li-Cor; 1:20,000).
9
RSV Protein Detection Using Western Blot and Immunostaining
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For western blot, the primary antibody used was a mouse monoclonal anti-RSV G (Abcam, Cat. No. ab94966) diluted to 2 µg/mL in Odyssey blocking buffer (LI-COR) with 0.1% Tween-20. The secondary antibodies were a donkey anti-mouse IRDye 680RD (LI-COR) and a donkey anti-rabbit IRDye 800 (LI-COR) and were diluted 1:3000 in Odyssey blocking buffer with 0.1% Tween-20.
For immunostaining, primary antibodies used were mouse anti-RSV N (Abcam, Cat. No. ab22501), human anti-RSV F (MedImmune, palivizumab), mouse anti-RSV F (Abcam, Cat. No. ab94968), mouse anti-RSV G (a gift from Ralph Tripp, clone 130-6D), rabbit anti-caveolin-1 (Santa Cruz, Cat. No. sc-894), and mouse anti-clathrin light chain (Biolegend, Cat. No. MMS-423P). The mouse anti-RSV F antibody was used only for the mRNA expression experiments in Fig.1 . All primary antibodies for immunostaining experiments were used at 1 µg/mL. Secondary antibodies used were donkey anti-rabbit Cy3 (Jackson ImmunoResearch), donkey anti-human Alexa Fluor 647, and donkey anti-mouse Alexa Fluor 488 (Life Technologies). All secondary antibodies for immunostaining experiments were used at 4 µg/mL.
For immunostaining, primary antibodies used were mouse anti-RSV N (Abcam, Cat. No. ab22501), human anti-RSV F (MedImmune, palivizumab), mouse anti-RSV F (Abcam, Cat. No. ab94968), mouse anti-RSV G (a gift from Ralph Tripp, clone 130-6D), rabbit anti-caveolin-1 (Santa Cruz, Cat. No. sc-894), and mouse anti-clathrin light chain (Biolegend, Cat. No. MMS-423P). The mouse anti-RSV F antibody was used only for the mRNA expression experiments in Fig.
10
Quantifying Protein Levels and Signaling Pathways
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Total protein concentration in whole cell lysates obtained using RIPA (Radio-ImmunoPrecipitation Assay Buffer), supplemented with protease and phosphatase inhibitor cocktail (Pierce™, Thermo Fisher Scientific, Waltham, USA), was determined using a BCA Protein Assay Kit according to the manufacturer’ s instructions (Pierce™, Thermo Fisher Scientific, Waltham, USA). Aliquots of cell extracts containing 20 μg of protein were resolved through SDS-PAGE. The proteins were subsequently transferred to a nitrocellulose membrane (Pierce™, Thermo Fisher Scientific, Waltham, USA). The membranes were hybridized with appropriate primary antibody: rabbit monoclonal phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), rabbit monoclonal p44/42 MAPK (ERK1/2) (137F5), rabbit monoclonal phospho-p70 S6 kinase (Thr389)(108D2), rabbit polyclonal p70 S6 kinase, rabbit monoclonal RSK1/RSK2/RSK3 (D7A2H), rabbit polyclonal phospho-Akt (S473), rabbit polyclonal Akt, rabbit monoclonal PI3 Kinase p110 δ (D1Q7R), rabbit monoclonal Histone H3 (3H1) and mouse monoclonal β-actin used as a loading control (Cell Signaling Technology, Danvers, USA). Detection of the specific proteins was performed directly on the nitrocellulose membranes using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, USA) and secondary antibody: donkey anti-rabbit IRDye 800 or donkey anti-mouse IRDye 680 (LI-COR Biosciences, Lincoln, USA).
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