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Irdye labeled secondary antibody

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IRDye-labeled secondary antibodies are fluorescently-tagged antibodies designed for use in various immunoassay techniques, such as Western blotting and immunohistochemistry. These antibodies are conjugated with near-infrared dyes that emit light when exposed to specific wavelengths, allowing for sensitive and quantitative detection of target proteins.

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39 protocols using irdye labeled secondary antibody

1

Analyzing PARP1 and Histone Acetylation

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A549 cells (10 cm dishes) were treated with vehicle or SBI-797812 (10 μM) for 4 h as described above. Where indicated, 0.5 mM H2O2 was also added to the cells for 30 min (from 3.5 to 4 h). Cell lysis buffer contained RIPA, NAM (10 mM), trichostatin-A (10 μg ml−1; Sigma-Aldrich), protease inhibitor cocktail (1×, Roche), olaparib (5 μM; Cayman Chemical), ADP-HPD (250 nM; Millipore Sigma) and benzonase nuclease (0.2 U μl−1; Sigma-Aldrich). Where indicated (Fig. 5c), olaparib and ADP-HPD were excluded during the cell lysis step. Samples were probed by western blotting with the following antibodies: rabbit monoclonal anti-PARP1 (Cell Signaling, cat no. 9532, clone 46D11) 1:1000 dilution, rabbit polyclonal (affinity-purified) anti-PAR (Trevigen 4336-APC-050) 1:1000-dilution, rabbit monoclonal anti-histone H4 (Cell Signaling, cat no. 13919, clone D2X4V) 1:1000-dilution, and rabbit polyclonal (affinity-purified) anti-acetyl-histone H4(Lys16) (Millipore Sigma, cat no. 07-329) 1:5000-dilution. Protein bands were visualized with IRDye-labeled secondary antibodies (LI-COR Biosciences) followed by scanning with the Odyssey Digital Infrared Imaging System (LI-COR Biosciences).
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2

Protein Extraction and Western Blot Analysis

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Protein was extracted with M-PER (Pierce, ThermoScientific, UK) for 30 min on ice in the presence of HALT protease inhibitor cocktail (Pierce). For phosphorylated proteins, cells were lysed in 1% hot SDS in Tris ph8.0 with phosSTOP phosphatase inhibitors (Roche) Proteins were separated by SDS-PAGE (4–12% acrylamide) and transferred to PVDF membranes (0.45um transfer membrane, ThermoFisher Scientific). Membranes were blocked with 5% (w/v) dried skimmed milk in TBS containing 0.5% Tween 20. Membranes were probed with anti-SINV, LC3 and actin antibodies as described. Proteins were detected with IRDye®-labeled secondary antibodies (Li-Cor Biosciences, Cambridge UK) at 1:10,000 dilution Proteins detected by the labelled secondary antibodies were visualized on the Odyssey infrared system
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3

Western Blot Analysis of Protein Samples

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After protein samples were denatured at 70°C for 10 min under reducing conditions, proteins were resolved with 4–12% gradient 1D-SDS-PAGE (Invitrogen, Carlsbad, CA), and transferred to 0.45 µm nitrocellulose (or PVDF) membranes (Bio-Rad, Hercules, CA) using a Bio-Rad semi-dry transblotter. The membranes were blocked in LiCor blocking buffer (LiCor, Lincoln, NE) and then incubated overnight at 4°C in LiCor blocking buffer with 0.1% Tween 20 and the primary antibodies listed in Table 2. All antibodies were individually optimized to determine ideal conditions within the linear range of detection for each assay, and that the primary antibody was present in excess. After antibody incubations, the membranes were rinsed in phosphate-buffered saline with 0.1% Tween (PBST) and probed with IR-dye labeled secondary antibodies (Li-Cor; 1:10,000) for 1 h at RT. The membranes were rinsed again with PBST and then with deionized water. Immunoblots were scanned using a LiCor Odyssey near-infrared scanner using the Odyssey V3.0.16 software package.
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4

Astrocyte and Neuron Culture Protocols

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Trans-cinnamic acid was obtained from Sigma Aldrich (C80857). Materials for primary astrocyte culture (DMEM/F-12, Hank’s balanced salt solution, 0.05% trypsin, and antibiotic-antimycotic) were obtained from Mediatech (Washington, DC). For primary neuron culture, neurobasal media, B27 supplement, B27 supplement minus antioxidant were purchased from Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Atlas Biologicals. Thioflavin-S and all other molecular biology-grade chemicals were purchased from Sigma Aldrich. All primary antibodies sources and dilutions used are listed in Table S1. Secondory antibodies (Alexa-fluor 488 and 647-conjugated) for immunostaining were purchased from Jackson ImmunoResearch. For immunoblotting, 10× Tris/Glycine/SDS running buffer was obtained from BioRad (1610772) and IR-dye-labeled secondary antibodies were obtained from Li-Cor Biosciences.
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5

Western Blot Protein Analysis Protocol

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Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40, protease inhibitor cocktail and phosphatase inhibitor cocktail) for 30 min on ice. Lysates were cleared by centrifugation at 12,000× g for 10 min at 4 °C. Samples were electrophoresed and transferred to polyvinylidene difluoride (PVDF) membranes. Blocking was conducted in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature, followed by overnight incubation of diluted primary antibodies. Membranes were further incubated with IRDye-labeled secondary antibodies (LI-COR) for 1 h at room temperature and scanned using Odyssey Infrared Imaging System (LI-COR).
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6

Western Blot Analysis of Cellular Proteins

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HEL cells were lysed in M-Per protein extraction reagent (Pierce Biotechnology, Waltham, MA) with protease inhibitor cocktail (Active Motif) and subjected to gel electrophoresis on 4% to 20% Mini-PROTEAN TGX gels (Bio-Rad) and the protein transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA). Blots were probed with antibodies against RAB31 (ARP61983_P050; Aviva Systems Biology, San Diego, CA), RUNX1 (sc-8563 or sc-365644), or actin (sc-1616R) or M6PR (sc-365196) from Santa Cruz Biotechnology, and mPR alpha polyclonal antibody (PA5-61376, Invitrogen/Thermo Fisher Scientific), and then with IRDye-labeled secondary antibodies (LI-COR Biosciences) using Odyssey Infrared Imaging System (LI-COR Biosciences).
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7

Protein Analysis of Brain Tissue

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Superior frontal cortex (Brodmann area 9) was dissected free of white matter at autopsy on dry ice to prevent thawing and was maintained at −80°C until assay. Tissue was homogenized (150 mg/ml) on ice in homogenization buffer (250 mM sucrose, 20 mM Tris base) containing protease and phosphatase inhibitors (Sigma), further diluted in homogenization buffer free of detergents or surfactants and analyzed for protein concentration with a NanoDrop (Thermo). For western blotting, 30 μg lysate was resolved on 8 or 10% Bis-Tris SDS polyacrylamide gels in a continuous buffer system and electrophoretically transferred to nitrocellulose membranes (BioRad) with a semi-dry blotter (Pierce) as described earlier [24 (link)–26 (link)]. Membranes were blocked for 1 h with blocking buffer (LI-COR Biosciences) and incubated in primary antibodies (Supplementary Table 1) overnight at 4°C. Membranes were then washed and incubated with IR-Dye-labeled secondary antibodies (1:18,000; LI-COR Biosciences) for 45 min at room temperature, washed again and visualized with the Odyssey infrared imaging system (LI-COR Biosciences). Blots were converted to binary, analyzed using ImageJ (NIH), and normalized to loading control (β-actin).
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8

Antibody-based Protein Detection Protocol

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Antibodies, their applications, sources and dilutions are listed in Supplementary Table 1. Cell culture materials (DMEMF/12, antibiotic/antimycotic) were purchased from Life Technologies. Original Ceylon cinnamon (Cinnamonum verum) in ground form was obtained from Indus Organics (San Ramon, CA). Other pharmacological compounds like sodium benzoate and sodium formate were purchased from Sigma-Aldrich. All molecular biology-grade and chemicals were obtained from Sigma or Bio-Rad. IR-Dye-labeled secondary antibodies used for immunoblotting were from Li-Cor Biosciences.
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9

Immunoblotting of c-MYC and SUMO Proteins

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Lysates were prepared from subconfluent cells harvested directly in boiling SDS lysis buffer (1% SDS, 11% glycerol, 10% β-mercaptoethanol, 0.1 M Tris pH 6.8) and boiled prior to SDS-PAGE. The following antibodies were used for detection: mouse monoclonal anti-c-MYC 9E10 (1∶1000, prepared in-house), rabbit polyclonal anti-c-MYC (1∶1000, Millipore #06-340), mouse monoclonal anti-Flag M2 (1∶1000, Sigma #3165), rabbit polyclonal anti-actin (1∶2500, Sigma #A2066), rabbit polyclonal SUMO1 (1∶1000, Abcam #ab32058), rabbit polyclonal SUMO2/3 (1∶1000, Abcam #ab3742), rabbit polyclonal anti-N-Myc (1∶500, Santa Cruz #sc-791). Primary antibodies were detected using IRDye-labeled secondary antibodies (1∶20000, LI-COR Biosciences) or HRP-conjugated secondary antibodies (1∶10000, GE Healthcare). For MG132 treatments, cells were treated with 10 µM MG132 (Calbiochem), diluted from a stock solution of 50 mM dissolved in DMSO for 4 hours prior to harvest.
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10

Western Blot Analysis of MAPK Signaling

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Frozen tissues were pulverized, and proteins were extracted in the modified RIPA buffer. Proteins from tissue or cell lysates were subjected to SDS‐PAGE and transferred to a nitrocellulose membrane followed by immunoblotting with the antibodies against phospho‐p38 MAPK‐Thr180/Tyr182, p38 MAPK, phospho‐JNK‐Thr183/Tyr185, JNK, phospho‐ERK‐Thr202/Tyr204, ERK (Cell Signaling Technology, CA), and GAPDH (Millipore, MA) or tubulin (Sigma, MA) for protein loading control. The blots were then incubated with IRDye labeled secondary antibodies (LI‐COR Biosciences, NE). The protein‐antibody complexes were visualized and quantified using the Odyssey Infrared Imaging System. Phosphorylation was calculated as a ratio of the phosphorylated protein to total protein and was then normalized to the untreated tissues (Ctrl) with the control value set as 1.0.
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