Primary antibodies include: 5-HT2CR detected by mouse monoclonal D-12 (sc-17797, Santa Cruz; 1:100); 5-HT2CR detected by goat polyclonal N-19 (sc-15081, Santa Cruz; 1:250); PLD1 detected by rabbit polyclonal H-160 (sc-25512, Santa Cruz; 1:100); PLD2 detected by rabbit polyclonal H-133 (sc-25513, Santa Cruz; 1:100); monoclonal mouse anti-b-actin (MAB1501, Chemicon International, Temecula, CA, 1:5000); monoclonal mouse anti pan-cadherin (C-19, Abcam, Cambridge, MA, 1:5000); Phospho-PLD1 (Thr147, pPLD1T147) (#3831, Cell Signaling Technology, Danvers, MA, 1:1000); Phospho-PLD1 (Ser561, pPLD1S561) (#3834, Cell Signaling Technology, Danvers, MA, 1:1000); Phospho-PLD2 (Phospho-Tyr169, pPLD2Y169) (A8400, Assay Biotech, Sunnyvale, CA, 1:1000). Secondary antibodies included infrared-labeled goat anti-mouse (IRDye 680; 926–32220, LI-COR Biosciences, Lincoln, NE); goat anti-rabbit (IRDye 800; 827–08365, LI-COR Biosciences); donkey anti-goat (IRDye 800CW; 605-731-125, Rockland Immunochemicals, Inc., Gilbertsville, PA) and sheep anti-mouse (IRDye 680, Rockland Immunochemicals).
Irdye 680
Manufactured by Rockland Immunochemicals
Sourced in Panama
IRDye 680 is a near-infrared fluorescent dye. It has an excitation wavelength of 680 nm and an emission wavelength of 700 nm. The dye can be used for a variety of applications, including cell and tissue imaging, protein labeling, and other fluorescence-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800, by Rockland Immunochemicals (4 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
H 160, by Santa Cruz Biotechnology (1 mentions)
H 133, by Santa Cruz Biotechnology (1 mentions)
Irdye 680, by LI COR (1 mentions)
Irdye 800, by LI COR (1 mentions)
Image studio digits, by LI COR (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
H 300, by Santa Cruz Biotechnology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Immobilion p, by Merck Group (1 mentions)
7 protocols using irdye 680
1
Antibody Validation for Serotonin and PLD
2
Baculovirus Protein Expression Analysis
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Baculoviruses were lysed with loading buffer containing 2% 2-mercaptoethanol, boiled for 5 min. The lysates were separated by 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes, and then probed with either of the following: an anti-PfCSP mouse mAb (2A10) or an anti-hDAF mouse mAb (anti-CD55, Merck Millipore, Temecula, CA), together with an anti-VP39 rabbit Ab [14 (link)]. Blots probed with the appropriate secondary Abs conjugated to IRDye 680 and IRDye 800 (Rockland Immunochemicals, Gilbertsville, PA) in the same membranes were visualized using an Odyssey infrared imager (LI-COR, Lincoln, NE). The molecular weight predictions were carried out on the ExPASy server, and densitometry analyses were performed using Image Studio Digits (LI-COR).
3
Alix Immunoblotting in EV and Soluble Proteins
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EV and soluble protein fractions were immunoblotted with anti-Alix primary antibody (3A9, Cell signaling), followed by secondary antibody conjugated with IRDye 680 (Rockland). Fluorescent signals were detected with an Odyssey infrared imaging system (LI-COR Biosciences).
4
Immunoblotting of Cellular and mRNP Proteins
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For immunoblotting of whole cell lysates and purified mRNPs, rabbit anti-UPF1 (H-300, Santa Cruz) and mouse anti-PABC1 (10E10, Abcam) were used. Secondary antibodies labeled with IRDye 680 or IrDye 800 (Rockland) were detected using an Odyssey imaging system (Li-Cor).
5
Quantitative Protein Analysis by Western Blot
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After performing specific treatments, cells were harvested and washed three times with PBS. After insoluble debris was pelleted by centrifugation at 15,000 g for 15 min at 4 °C, the supernatants were collected and examined for protein concentrations using the bicinchoninic acid (BCA) kit (Pierce, USA) according to the manufacturer’s instructions. Proteins were separated by electrophoresis on SDS-polyacrylamide gel electrophoresis (PAGE) and electro-transferred to nitrocellulose filter (NC) membrane. After being blocked for 2 h in 5% bovine serum albumin (BSA), membranes were incubated overnight at 4 °C with the following primary antibodies: anti-Rap2B, anti-p-ERK1/2, anti-ERK1/2 and anti-β-actin. Membranes were then washed and incubated with secondary antibodies conjugated with IRDye 680 or IRDye 800 (Rockland). Fluorescent signals were visualized with Odyssey infrared imaging system (Li-COR Lincoln, NE).
6
Quantitative Staphylococcal Antigen Profiling
7
Western Blot Analysis of Protein Samples
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For each sample equal amounts of total protein (12 μg) mixed with 5x loading buffer (153 mM TRIS pH = 6.8, 7.5% SDS, 40% glycerol, 5 mM EDTA, 12.5% 2-β-mercaptoethanol and 0.025% bromophenol blue) were loaded into 10% SDS-polyacrylamide gels, and transferred to a PVDF membrane (Immobilion-P, Millipore, Cat# IPFL00005). 5% BSA in Tris-buffered saline (TBS) (100 mmol/L NaCl, 10 mmol/L Tris, pH = 7.4) and 0.1% Tween-20 was used to block membranes for 1 hr and then immunoblotting of the membranes was performed overnight at 4°C by incubating with the primary antibodies listed in Table 3 . Finally, a 1 hr incubation of the membranes with their respective secondary fluorescent antibodies: anti-mouse (1:2500, IRDye 800, Rockland, Cat# 610-132-121) and anti-rabbit (1:2500, IRDye 680, Rockland, Cat# 611-144-002) was performed. Immunoreactive bands were detected using a LI-COR Odyssey scanner (LI-COR Biosciences). Normalization of protein densities compared to the expression signal of the housekeeping gene in the same samples was performed and expressed relative to the P7 time-point.
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