Ir800

Manufactured by LI COR

Sourced in United States

The IR800 is a near-infrared imaging system designed for scientific research applications. It utilizes infrared light to capture high-quality images and data. The core function of the IR800 is to provide non-invasive, real-time imaging and analysis capabilities for a variety of scientific and industrial applications.

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Lab products found in correlation

Ir680, by LI COR (5 mentions) Odyssey infrared imaging system, by LI COR (3 mentions) Ir700, by LI COR (3 mentions) Odyssey blocking buffer, by LI COR (2 mentions) Bca assay, by Thermo Fisher Scientific (2 mentions) Rabbit anti ha antibody, by Cell Signaling Technology (1 mentions) Tunel staining kit, by Roche (1 mentions) Protein assay dye reagent, by Bio-Rad (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Gadph, by Santa Cruz Biotechnology (1 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions) Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Odyssey scanner, by LI COR (1 mentions) Ab6046, by Abcam (1 mentions) Clone 4a6, by Merck Group (1 mentions) Ab157106, by Abcam (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Ab8226, by Abcam (1 mentions) Sc 48794, by Santa Cruz Biotechnology (1 mentions) Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)

Spelling variants of this product

IR800 dye (6 citations) Secondary goat-anti rabbit-IR800 (3 citations) Maleimide-IR800 probe (3 citations) Streptavidin-IR800 (2 citations) IR800 label (2 citations) IR800 dye conjugated anti-mouse IgG (2 citations) Goat anti-mouse IgG IR800 Dye 800 CW (2 citations) IR800 dye-maleimide probe (2 citations) α-rabbit IR800/α-mouse IR680 secondary antibodies (2 citations) Goat anti-rabbit IgG IR800 Dye 800 CW (2 citations)

13 protocols using ir800

Molecular Toolkit for RELT Pathway Investigation

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The human embryonic kidney 293 (293) cell line was purchased from ATCC.
The mammalian expression plasmid constructs for RELT, RELL1, RELL2, RFRV mutant
of RELT [8 (link)] and MACH 360
C/S [21 (link)] were described
previously. Expression constructs for TRAF2, TRAF2-RING, CrmA and Fadd DN were a
kind gift from D. Goeddel. The MEKK3 plasmid was a kind gift from G. Johnson.
The human tissue extracts, monoclonal anti-RELT (C-6), p38 alpha/beta (A-12) and
GADPH antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,
CA). The RELT polyclonal antibody (AF1385) was purchased from R&D systems
(Minneapolis MN). The phospho-p38 (D3F9), mouse anti-DYKDDDDK (equivalent to
FLAG) tag and rabbit anti-HA antibody were purchased from Cell Signaling
Technology, Inc. (Danvers, MA). IR-800 and IR-680 secondary antibodies were
purchased from LI-COR Biosciences (Lincoln, NE). TUNEL staining kit was
purchased from Roche Diagnostics (Mannheim, Germany). The CHOP luciferase
trans-reporting system and QuickChange site-directed mutagenesis kit were
purchased from Stratagene (La Jolla, CA).

Moua P., Checketts M., Xu L.G., Shu H.B., Reyland M.E, & Cusick J.K. (2017). RELT family members activate p38 and induce apoptosis by a mechanism distinct from TNFR1. Biochemical and biophysical research communications, 491(1), 25-32.

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Western Blot Analysis of TRPM7, MycN, and ODC

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Cell lysates were prepared in RIPA buffer [20 mM Tris–HCl, pH 7.5, 0.1% (w/v) sodium lauryl sulfate, 0.5% (w/v) sodium deoxycholate, 135 mM NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mM EDTA], supplemented with Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA), and phosphatase inhibitors sodium fluoride (NaF) (20 mM) and sodium vanadate (Na₃VO₄) (0.27 mM). Western blot analysis was performed as previously described [28] (link). The total protein concentration was determined using the protein assay dye reagent from Bio-Rad Laboratories (Hercules, CA, USA). Cell lysates in SDS-sample buffer were boiled for 5 min and equal amounts of total protein analyzed by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting. The antibodies used in this study are: rabbit monoclonal TRPM7 (at a 1:1000 dilution from Epitomics, Inc. (Burlingame, CA, USA)), rabbit polyclonal MycN (at a 1:500 dilution), goat polyclonal ODC (at a 1:500 dilution) and mouse monoclonal GADPH (at a 1:1000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary anti-mouse (at a 1:10,000 dilution) IR-680 or IR-800 (LI-COR Biosciences, Lincoln, NB, USA). Proteins were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NB, USA) and analyzed with Licor Image Studio 2.0 acquisition and analysis software.

Lange I, & Koomoa D.L. (2014). MycN promotes TRPM7 expression and cell migration in neuroblastoma through a process that involves polyamines. FEBS Open Bio, 4, 966-975.

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Immunoblotting Procedure for Protein Expression

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Immunoblotting was performed as described (60 (link)). Briefly, cultured cells were lysed using radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Roche). Proteins were resolved with SDS–polyacrylamide gel electrophoresis, and after transfer to a nitrocellulose membrane were blocked in Odyssey blocking buffer (LI-COR). The following primary antibodies were used: human NAF1 (rabbit, ab157106, 1:1000; Abcam), mouse Naf1 (rabbit, Naf1 394, 1:250; Prosci), Myc (mouse, clone 4A6, 1:1000; Millipore), human dyskerin (rabbit, sc-48794, 1:250; Santa Cruz Biotechnology), and green fluorescent protein (mouse, 7.1 and 13.1, 1:1000; Roche) with loading controls actin (mouse, ab8226, 1:2000; Abcam), tubulin (rabbit, ab6046, 1:5000; Abcam), PARP (rabbit, 9542S, 1:1000; Cell Signaling Technology), or GAPDH (mouse, mAbcam9498, 1:1000; Abcam). Secondary antibodies were conjugated to dyes IR680 or IR800 (donkey, 1:10,000; LI-COR), and blots were visualized using an Odyssey scanner (LI-COR), with exception of the mouse Naf1 antibody that was visualized by horseradish peroxidase–linked antibody (rabbit, 7074, 1:10,000; Cell Signaling Technology) and enhanced chemiluminescent substrate (Thermo Scientific). Cell fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific).

Stanley S.E., Gable D.L., Wagner C.L., Carlile T.M., Hanumanthu V.S., Podlevsky J.D., Khalil S.E., DeZern A.E., Rojas-Duran M.F., Applegate C.D., Alder J.K., Parry E.M., Gilbert W.V, & Armanios M. (2016). Loss-of-function mutations in the RNA biogenesis factor NAF1 predispose to pulmonary fibrosis–emphysema. Science translational medicine, 8(351), 351ra107.

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Protein Quantification and Immunoblotting

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RIPA (Sigma) was used to prepare total lysate. Fractionation method was performed as previously described [55 (link)]. Bicinchoninic Acid (BCA) assay (Thermo Scientific) was used to determine protein concentration. Equal amounts of lysates were subjected to immunoblotting on SDS-PAGE. IRDyes IR680, IR680-LT, or IR800 (LI-COR) were used as secondary antibodies. LI-COR Odyssey 2.1 system was used to visualise proteins. 16-bit images were analyzed and quantified using Image J.

Faronato M., Nguyen V.T., Patten D.K., Lombardo Y., Steel J.H., Patel N., Woodley L., Shousha S., Pruneri G., Coombes R.C, & Magnani L. (2015). DMXL2 drives epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through notch hyper-activation. Oncotarget, 6(26), 22467-22479.

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Cell Lysis and Protein Analysis

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For protein analysis, cleared NP40 lysates and whole cell SDS lysates were prepared from 10-cm dishes containing cells at 100% confluency as described [17 (link), 26 (link)]. Protein concentration was measured by BCA assay and 50 micrograms protein was loaded per lane. Immunoprecipitations were performed as described except that the buffer contained 0.5% NP40 [11 (link)]. Antibodies for E-cadherin, N-cadherin, beta catenin, phospho-beta catenin, beta four integrin, and fibronectin were all rabbit polyclonal from Cell Signaling; for Myc-tag (clone 9E10.3), from Neomarkers; for FLAG tag (clone M2), Agilent; for actin (125-ACT), from PhosphoSolutions; for GFP, from Abcam; for vimentin, from Millipore. ZO-1 antibody was from PTG. The CLCA2 antibody TVE20 has been described (12). The protein size marker was Dual color (Bio-Rad). Secondary antibodies were labeled with IR680 or IR800 (Licor), and protein expression was quantified on an Odyssey instrument (Licor).

Ramena G., Yin Y., Yu Y., Walia V, & Elble R.C. (2016). CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation. PLoS ONE, 11(3), e0147489.

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Quantification of alpha-synuclein in mouse brain

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Brains from male and female mice 3–4 months of age were dissected immediately postmortem, and olfactory bulbs and cerebellum were removed. Remaining brain tissue was prepared for western blot analysis via use of Syn-PER Synaptic Protein Extraction Reagent (Thermo Fisher) using the manufacturer’s protocol, resulting in both a synaptosomal and a cytosolic fraction. Samples were run on a 4–12% Tris-glycine gels (Invitrogen) for 2 h and 45 min at 80 V. Gels were transferred onto Immobilon-FL membranes (0.45 μm pore; Millipore) at 25 V for 18 h at 4 °C. Membranes were blocked with Odyssey blocking buffer (Li-COR Biosciences) for 1 h at room temperature. Membranes were incubated overnight with primary antibodies (alpha-synuclein: 1:1000, Syn-1, BD Biosciences; serine-129 phospho-alpha-synuclein: 1:1000, EP1536Y, Abcam; GAPDH: GAPDH, 1:10,000, Millipore). A near infrared fluorescent-labeled secondary antibodies (1:5000; IR800 & IR700; LI-COR Biosciences) were used, and quantification was done with an Odyssey CLx infrared imaging system (LI-COR Biosciences) and ImageJ/Fiji (NIH).

Weston L.J., Stackhouse T.L., Spinelli K.J., Boutros S.W., Rose E.P., Osterberg V.R., Luk K.C., Raber J., Weissman T.A, & Unni V.K. (2021). Genetic deletion of Polo-like kinase 2 reduces alpha-synuclein serine-129 phosphorylation in presynaptic terminals but not Lewy bodies. The Journal of Biological Chemistry, 296, 100273.

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Fly Head Protein Extraction and Western Blot

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Proteins were extracted from fly heads with a homogenizing buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulphonyl fluoride (PMSF)). Samples were spun at 14,000g for 30 min at 4 °C. Supernatant 1 was collected for later analysis. The pellets were resuspended with a protein extraction buffer (30 mM HEPES pH 7.9, 0.6 M NaCl, 1.5 mM MgCl2, 0.4 mM EDTA, 1 mM DTT, 25% glycerol, 1% NP-40 and 0.5 mM PMSF) and incubated at 4 °C for 30 min with vortexing every 6 min. Then the supernatant 2 was collected after 30 min centrifugation at 14,000g at 4 °C. Next, supernatants 1 and 2 were equally mixed and heated at 95 °C with 4 × Laemmli sample buffer. Lysates were probed with anti-DmNMNAT (1:8,000); Living Colors AcGFP Monoclonal Antibody (JL-8; 1:1,000, Clontech, Mountain View, CA, USA); Living Colors DsRed Polyclonal Antibody (1:1,000, Clontech); anti-cleaved caspase 3 (1:1,000, Cell Signaling, Beverly, MA, USA) and anti-β-Actin antibody (1:10,000, Sigma). Western blot analysis was carried out using the infrared dye-conjugated secondary antibodies, IR700 and IR800 (LI-COR Biosciences), and blots were imaged and processed on an Odyssey Infrared Imaging system.

Ruan K., Zhu Y., Li C., Brazill J.M, & Zhai R.G. (2015). Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress. Nature Communications, 6, 10057.

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GRK2 Protein Immunoblotting Protocol

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Membranes were blocked in 5% milk in Tris-buffered saline/0.1%Tween 20 (TBST) for 2 h at room temperature. After three washes (5 min each) with TBST, the membranes were incubated with anti-GRK2 antibody (1:3000) in 3% bovine serum albumin (BSA) in TBST overnight at 4°C. After incubation, membranes were washed four times with TBST (10 min each) and incubated with anti-rabbit secondary antibody (1:10,000, IR-800; LI-COR Biosciences, Lincoln, NE) in 5% milk in TBST for 1 h at room temperature. Blocking was for 1 h at room temperature, and primary antibody was incubated for 2 h at room temperature and secondary antibody for 1 h at room temperature. The membranes were washed three times with TBST (10 min each) and scanned on a LI-COR Odyssey infrared imaging system.

Malik S., deRubio R.G., Trembley M., Irannejad R., Wedegaertner P.B, & Smrcka A.V. (2015). G protein βγ subunits regulate cardiomyocyte hypertrophy through a perinuclear Golgi phosphatidylinositol 4-phosphate hydrolysis pathway. Molecular Biology of the Cell, 26(6), 1188-1198.

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Cortical Protein Analysis in Mice

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Mice were sacrificed at 1.5, 3 and 6 months of age (n = 3 per genotype and age). The cortex was dissected and snap frozen and kept at − 80 °C until use. For protein extraction, tissues were homogenized in lysis buffer consisting of 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, protease inhibitor cocktail (Sigma, St Louis, MO, USA) and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Total protein concentration was measured using the Bradford Reagent (Thermo Scientific/Pierce Biotechnology, Rockford, IL, USA).
Ten micrograms of total protein lysate per sample was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane, and probed with antibodies against GFAP (1/4000; DAKO, Carpinteria, CA, USA), neurofilament medium (NF-M, 1/4000; Encore, Gainesville, FA, USA), GAPDH (1/5000; Sigma), hTAU (1/1000; DAKO), PHF-1 (1:1000, gift from Peter Davis), AT8 (1:1000, Pierce), and pSer262 tau (1:2000, Millipore). Western blot analysis was performed with infrared dye conjugated secondary antibodies, IR700 and IR800 (LI-COR Biosciences, Lincoln, NE, USA). Blots were imaged and processed on an Odyssey® Infrared Imaging System.

Majid T., Ali Y.O., Venkitaramani D.V., Jang M.K., Lu H.C, & Pautler R.G. (2014). In vivo axonal transport deficits in a mouse model of fronto-temporal dementia. NeuroImage : Clinical, 4, 711-717.

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4-HNE Protein Adducts Analysis

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Protein from frozen striatum and midbrain was extracted with 1 × radioimmunoprecipitation assay buffer (CST, Beverly, MA, USA) with 1 × Calbiochem Protease Inhibitor Cocktail Set I and 1 × Halt* Phosphatase Inhibitor Cocktail (Thermo Scientific). A Tissue Lyser LT (Invitrogen, Pleasanton, CA, USA) with a 5 mm steel bead was used to homogenize tissues at 50 Hz. Tissue lysate was then centrifuged at 13,000 rpm for 15 min at 4°C. Supernatant was saved in a –80°C freezer. Bradford protein assay reagents (Bio-Rad, Irvine, CA, USA) were used to determine protein concentration. Protein (40 μg/lane) was then separated on a 4–12% Criterion sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (Bio-Rad) and transferred on to nitrocellulose membrane. Proteins were detected with antibodies directed specifically to 4-HNE protein adducts (R&D, Minneapolis, MN, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST), respectively, in the midbrain and striatum of 6- and 18-month-old mice. An appropriate secondary antibody from LiCor (IR800 or IR680) was used corresponding to each primary antibody. Immunoreactive bands were imaged and quantified using Odyssey software (LiCor, Lincoln, NE, USA).

Bai X., Wey M.C., Martinez P.A., Shi C., Fernandez E, & Strong R. (2017). Neurochemical and motor changes in mice with combined mutations linked to Parkinson’s disease. Pathobiology of Aging & Age Related Diseases, 7(1), 1267855.

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