Goat anti mouse irdye 680lt secondary antibody

Manufactured by LI COR

Sourced in United States

The Goat anti-mouse IRDye 680LT secondary antibody is a fluorescently labeled secondary antibody designed for use in western blotting, immunohistochemistry, and other immunoassay applications. The antibody binds to mouse primary antibodies and is conjugated to the IRDye 680LT fluorescent dye, allowing for detection and quantification of target proteins.

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Lab products found in correlation

Irdye 800cw goat anti rabbit, by LI COR (4 mentions) Irdye 800cw goat anti rabbit secondary antibody, by LI COR (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Ripa lysis buffer, by Merck Group (2 mentions) Irdye 800cw donkey anti rabbit, by LI COR (1 mentions) Quantitative fluorescent imaging system odyssey, by LI COR (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Odyssey system, by LI COR (1 mentions) β actin primary antibody, by Merck Group (1 mentions) Mglur2, by Abcam (1 mentions) H3k9ac, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) β actin antibody, by Cell Signaling Technology (1 mentions) Mini gels, by Thermo Fisher Scientific (1 mentions) Newblot nitro stripping buffer, by LI COR (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Mouse monoclonal antibody to human β actin, by Merck Group (1 mentions) Complete protease inhibitor cocktail tablet, by Roche (1 mentions) Odyssey v3, by LI COR (1 mentions)

8 protocols using goat anti mouse irdye 680lt secondary antibody

Quantification of Spy Variants and Biosensor

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The steady-state expression levels of the different Spy variants and the β-lactamase-Im7 L53A I54A biosensor in strains LW53-LW59 (Table 1) were quantified by western blotting of whole cell extractions using infrared fluorescence labeled IRDye secondary antibodies. This was done after induction by various amounts of IPTG (0.01–0.5 mM) for 3 hr. IRDye 680LT goat anti-mouse secondary antibody (LI-COR Biosciences, Lincoln, NE) was used to recognize the primary antibodies, which were against either the β-lactamase portion of the biosensor or directed against Spy. As a loading control, we quantified the amount of maltose binding protein (MBP) present in the lysate using a IRDye 800CW goat anti-rabbit secondary antibody (LI-COR Biosciences) directed against MBP. 0.04% maltose was added to the medium to induce MBP expression. MBP is a periplasmic protein that is routinely used as a loading control (Raivio et al., 1999 (link)); it has the added advantage that it is not a Spy client (unpublished results). Various dilutions of the whole cell extracts (A₆₀₀ = 2.5) were loaded and their intensities were plotted to quantify the level of Spy and β-lactamase-Im7 L53A I54A biosensor protein in the Spy variants.

Quan S., Wang L., Petrotchenko E.V., Makepeace K.A., Horowitz S., Yang J., Zhang Y., Borchers C.H, & Bardwell J.C. (2014). Super Spy variants implicate flexibility in chaperone action. eLife, 3, e01584.

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Western Blot Analysis of Lpd in HeLa Cells

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Western blot analyses were performed using cytoplasmic extracts subjected to SDS-PAGE. Confluent monolayers of HeLa cells were washed twice with PBS and subsequently scraped in a 250-μl volume of ice-cold NP-40 buffer containing 50 mM Tris-HCl, 200 nM NaCl, 1% Triton X-100, 2 mM MgCl₂, 10% glycerol, and a 1/20 final volume of protease inhibitor cocktail (Sigma). Following 15 min of incubation on ice, cell lysates were cleared by centrifugation at 16,000 × g at 4°C and supernatants were subjected to SDS-PAGE using an 8% polyacrylamide gel and transferred to a nitrocellulose membrane. Lpd was probed with rabbit anti-LPD serum (F. B. Gertler, MIT, USA) at 1:1,000 dilution and IRDye 800CW donkey anti-rabbit at 1:7,500 dilution (Li-COR Biosciences). Monoclonal anti-α-tubulin raised in mouse (Sigma) at 1:1,000 dilution and IRDye 680LT goat anti-mouse secondary antibody (Li-COR Biosciences) at 1:10,000 dilution were used as controls. Images were developed using an Odyssey quantitative fluorescent imaging system (Li-COR Biosciences).

Wang J., King J.E., Goldrick M., Lowe M., Gertler F.B, & Roberts I.S. (2015). Lamellipodin Is Important for Cell-to-Cell Spread and Actin-Based Motility in Listeria monocytogenes. Infection and Immunity, 83(9), 3740-3748.

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Histone H3 Acetylation and mGluR2 Analysis

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Tissues were homogenized in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) supplemented with 1 mM Na₃VO₄ and protease inhibitor cocktail (Roche, Branford, CT, USA). The homogenates were centrifuged at 14,000×g for 10 min at 4°C and the supernatant was collected. Protein contents in supernatants were measured using the Bradford method. After denaturing protein samples were fractionated 25 µg per lane on 4–12% SDS-NuPAGE gel and blotted to nitrocellulose membrane. The membranes were incubated with primary antibody (1:1,000) directed against acetylated lysine 9 on histone 3 (H3K9ac, Cell Signaling Technology, Danvers, MA, USA) or mGluR2 (Abcam Inc, Cambridge, MA, USA) at 4°C overnight. The membranes were further incubated for 1 hour in blocking buffer with IRDye 800CW goat anti-rabbit secondary antibody (Li-Cor Biosciences, Lincoln, NE, USA). Washed membranes were scanned and analyzed with the Li-Cor Odyssey System (Li-Cor Biosciences, Lincoln, NE, USA). Blots were then stripped for 30 min and reprobed with antibodies for pan-histone 3 (1:1000, Cell Signaling Technology) or β-actin primary antibody (1:5,000, Sigma, St. Louis, MO, USA) and IRDye 800CW goat anti-rabbit secondary antibody or IRDye 680LT goat anti-mouse secondary antibody (1:20,000, Li-Cor Biosciences) with same processes as above.

Cao D.Y., Bai G., Ji Y, & Traub R.J. (2014). Epigenetic upregulation of metabotropic glutamate receptor 2 in the spinal cord attenuates estrogen-induced visceral hypersensitivity. Gut, 64(12), 1913-1920.

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Quantitative western blotting analysis

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Transfected cell pellets were re-suspended (50 μl, 10 min) in detergent lysis buffer (1% IGEPAL, 50 mM Tris-HCL, 150 mM NaCl, pH 7.8) containing HALT protease inhibitor (Pierce/Thermo Scientific, Rockford, IL) then centrifuged (10,000 x g, 10 min) to pellet cell nuclei. Post-nuclear lysate was removed and soluble protein concentration was determined using the Non-interfering assay (G-Bioscience, St. Louis, MO). Soluble proteins (10 μg) and molecular weight markers (10–250 kDa, Li-Cor, Lincoln, NE) were separated on SDS-PAGE gels (10% mini gels, Invitrogen/Life Technologies) then transferred onto nitrocellulose, incubated with FLAG primary antibody (anti-D 1:1000 dilution, GeneCopoeia) followed by goat-anti-mouse IRDye 680LT secondary antibody (1:10,000 dilution, Li-Cor). Protein bands were visualized using an Odyssey Infrared Imaging System (Li-Cor) running Image Studio (Ver 4.0) software. Blots were stripped (NewBlot nitro stripping buffer, Li-Cor) and re-probed with β-actin antibody (1:1000 dilution, Cell Signaling, Danvers, MA) to control for sample-loading and quantification of protein band intensity.

Mackay D.S., Bennett T.M, & Shiels A. (2015). Exome Sequencing Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with Autosomal Dominant Primary Open-Angle Glaucoma. PLoS ONE, 10(7), e0132529.

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Quantitative Western Blot Analysis of SOX9

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Protein lysates of the cells were extracted with 1× RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA) supplemented with complete protease inhibitor cocktail tablets (Roche Applied Science, IN, USA) after incubation with SOX9 siRNA/non-specific siRNA for 48 hours. Western blotting was performed as follows: denatured proteins were run on NuPAGE^® 4–12% Bis-Tris Gel (Life Technologies), and then transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked in 5% nonfat milk for 1 hour, and incubated with specific primary antibody (Sox-9 (H-90): sc-20095, Santa Cruz Biotechnology, 1:1000 dilution) or mouse monoclonal antibody to human β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight. Following primary antibody incubation, membranes were washed with PBST (1×), and goat anti-rabbit IRDye^® 800CW or goat anti-mouse IRDye^® 680LT secondary antibody (1:20000 dilution) (926-32211 and 926-68020, Li-COR Biosciences, NE, USA) were added, respectively. Bands were detected using Odyssey for Infrared Fluorescent Western Blots from Li-COR Bioscience (Lincoln, NE, USA). Quantification analysis of Western blot bands was performed with ImageJ software (National Institutes of Health, USA). All primary antibodies used in this study are described in Supplementary Table 3. The Western blot assay was conducted in duplicate.

Chen H., Garbutt C.C., Spentzos D., Choy E., Hornicek F.J, & Duan Z. (2017). Expression and Therapeutic Potential of SOX9 in Chordoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(17), 5176-5186.

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Western Blot Protein Quantification

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Total protein lysate was extracted from cells using RIPA lysis buffer (Sigma-Aldrich; Merck KGaA). Thirty micrograms of the protein sample was separated on NuPage 4–12% Bis-Tris gel (Thermo Fisher Scientific, Inc.) and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.), and then incubated with the following primary antibodies in 5% milk with tris-buffered saline Tween 20: CDK9 (cat. no. 2316; 1:1,000 dilution; Cell Signaling Technology, Inc.), Mcl-1 (cat. no. 39224; 1:1,000 dilution; Cell Signaling Technology, Inc.), PARP (cat. no. 9532; 1:1,000 dilution; Cell Signaling Technology, Inc.), Bax (cat. no. 2772; 1:1,000 dilution; Cell Signaling Technology, Inc.) and tubulin (cat. no. 3873; 1:1,000 dilution; Cell Signaling Technology, Inc.). Next, the membranes were further incubated with a goat anti-rabbit IRDye 800CW (926–32,211; 1:5,000 dilution; Li-Cor Biosciences) or goat anti-mouse IRDye 680LT secondary antibody (926–68,020; 1:15,000 dilution; Li-Cor Biosciences) at room temperature for 2 h. The membranes were then scanned by an Odyssey CLx device (Li-Cor Biosciences) to detect bands. Finally, protein bands were quantified by densitometry with Odyssey v. 3.0 software (Li-Cor Biosciences).

He S., Fang X., Xia X., Hou T, & Zhang T. (2020). Targeting CDK9: A novel biomarker in the treatment of endometrial cancer. Oncology Reports, 44(5), 1929-1938.

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Protein Extraction and Quantification for Western Blot

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The protein was extracted from cells and tissues by 1× RIPA lysis buffer (Sigma-Aldrich) combined with protease inhibitor cocktail tablets (Roche Applied Science, IN). The protein lysate concentrations were then determined using the DC protein assay reagents (Bio-Rad, CA) and a spectrophotometer SPECTRA max 340PC (Molecular Devices, LLC, CA). Western blot was performed using similar methods to those previously described. 23 In short, an SDS-PAGE gel was used to run the denatured proteins before they incubation with the primary antibody, TBST was then used as a membrane wash (three times, 5 minutes, room temperature).
Next, goat anti-rabbit IRDye 800CW (926-32,211, 1:5000 dilution) or goat anti-mouse IRDye 680LT secondary antibody (926-68,020, 1:15 000 dilution) (Li-COR Biosciences) was applied for 2 hours at room temperature followed by another TBST membrane wash (three times, 5 minutes, room temperature). Bands were detected using an Odyssey Infrared Fluorescent Western Blots Imaging System from Li-COR Bioscience and Odyssey software 3.0 was used to quantify the bands.

Wei R., Thanindratarn P., Dean D.C., Hornicek F.J., Guo W, & Duan Z. (2020). Cyclin E1 is a prognostic biomarker and potential therapeutic target in osteosarcoma. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 38(9).

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Western Blot Analysis of Chordoma Proteins

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Cell pellets and human chordoma tissues were lysed in protein lysis buffer. The protein concentrations were calculated with Protein Assay Reagents (Bio-Rad, CA) and a SpectraMax 340PC Microplate Reader from Molecular Devices (San Jose, CA). Equal amounts of protein were separated on 4-12% Bis-Tris gels (NuPAGE®, Thermo Fisher Scientific, CA) and transferred to nitrocellulose membranes, where they were incubated with specific primary antibodies at 4°C overnight (CDK9 1:1000 dilution; RNAP II at 1: 1000 dilution; p-RNAP II Ser-2 at 1: 1000 dilution; Mcl-1 at 1: 1000 dilution; Bax at 1: 1000 dilution; Survivin at 1: 1000 dilution α -Tubulin at 1: 1000 dilution). After being washed with TBST three separate times for five minutes, the membranes were further incubated with Goat anti-rabbit IRDye 800CW or Goat anti-mouse IRDye 680LT secondary antibody (LI-COR Biosciences, NE. Final images were obtained with Odyssey® CLx equipment (LI-COR Biosciences). The abundance of α-Tubulin was monitored to ensure equal loading.

Shen S., Dean D.C., Yu Z., Hornicek F., Kan Q, & Duan Z. (2020). Aberrant CDK9 expression within chordoma tissues and the therapeutic potential of a selective CDK9 inhibitor LDC000067. Journal of Cancer, 11(1), 132-141.

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