Immunoprecipitation and immunoblotting analyses were performed as previously described.40 (link) The following secondary antibodies were used, goat anti-mouse 800CW, goat anti-rabbit 800CW, goat anti-mouse 680LT or goat anti-rabbit 680 (all from LI-COR Biosciences GmbH, Lincoln, NE, USA). The membranes were scanned using Odyssey Imager from LI-COR Biosciences GmbH.
Goat anti mouse 680lt
Manufactured by LI COR
Sourced in United States
The Goat anti-mouse 680LT is a secondary antibody product designed for use in fluorescence-based detection applications. It is conjugated with the 680LT dye, which has an excitation wavelength of 680 nm and an emission wavelength of 700 nm. This product can be used to detect and quantify target proteins or other biomolecules in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit 800cw, by LI COR (11 mentions)
Goat anti mouse 800cw, by LI COR (6 mentions)
Anti β actin clone ac 15 a5441, by Merck Group (4 mentions)
Odyssey clx imaging system, by LI COR (4 mentions)
Odyssey imager, by LI COR (3 mentions)
Anti fak clone 77 610088, by BD (3 mentions)
Rhodamine labeled phalloidin, by Thermo Fisher Scientific (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Mini protean iitm system, by Bio-Rad (1 mentions)
Goat anti human irdye 800 cw, by LI COR (1 mentions)
Anti py397 fak, by Thermo Fisher Scientific (1 mentions)
Anti phosphotyrosine clone 4g10 05 321, by Merck Group (1 mentions)
Alexa fluor conjugated secondary, by Thermo Fisher Scientific (1 mentions)
Anti cortactin ab 33333, by Abcam (1 mentions)
Anti arp2 h 84 sc 15389, by Santa Cruz Biotechnology (1 mentions)
Anti tks5 fish m 300 sc 30122, by Santa Cruz Biotechnology (1 mentions)
Anti gfp clones 7 1 and 13, by Roche (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Ab137446, by Abcam (1 mentions)
18 protocols using goat anti mouse 680lt
1
Immunoprecipitation and Immunoblotting Protocol
2
Infrared Fluorescent Western Blotting for GA^P and CO^P
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Infrared fluorescent western blotting was conducted to confirm the expression of both GAP and COP in plants. Purified recombinant proteins mixed with 4 μL of 5× loading buffer were electrophoresed using 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA) using the Mini-Protean IITM system (Bio-Rad, Hercules, CA, USA). Membranes were incubated in TBS buffer for 4 h at RT, followed by incubation with the goat anti-human IRDye 800 CW and goat anti-mouse 680 LT (1:15,000; LI-COR, Lincoln, NE, USA) in the blocking buffer at RT for 1.5 h to detect GAP and COP, respectively. After washing four times for 5 min. each in 1 × TBS at RT, the membranes were scanned using the OdysseyTM CLx infrared imaging system (LI-COR).
3
Immunofluorescence and Western Blotting Assay
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For immunofluorescence, anti-cortactin (ab-33333) was obtained from Abcam; anti–pY402-Pyk2 and anti–pY397-FAK were obtained from Invitrogen; anti-Arp2 (H-84; SC-15389) and anti-Tks5 (FISH M-300; SC-30122) were obtained from Santa Cruz Biotechnology, Inc.; and antivinculin (clone hVIN1; V9131), anti–pY421-cortactin (C0739), and anti–pY466-cortactin (C0864) were obtained from Sigma-Aldrich. Rhodamine-labeled phalloidin and Alexa Fluor–conjugated secondary antibodies were obtained from Molecular Probes. For Western blotting, anticortactin (clone 4F11; 05-180) and antiphosphotyrosine (clone 4G10; 05-321) were obtained from EMD Millipore; anti-Pyk2 (3480 and 3292) was obtained from Cell Signaling Technology; anti-FAK (clone 77; 610088) was obtained from BD; anti-GFP (clones 7.1 and 13.1; 11814460001) was obtained from Roche; and anti–β-actin (clone AC-15; A5441) was obtained from Sigma-Aldrich. Secondary antibodies (goat anti–mouse 680LT and goat anti–rabbit 800CW) were obtained from LI-COR Biosciences.
4
Immunodetection and Immunostaining Protocols
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The following primary and secondary antibodies were applied for immunodetection and immunostaining: anti-ataxin-3 (1:5000; 1H9, MAB5360, Merck, Darmstadt, Germany), anti-ataxin-3/C-terminal (1:2500; SA3637) [20 (link)], anti-KPNB1 (1:5000; H-300, sc-11367, Santa Cruz Biotechnology, Heidelberg, Germany), anti-KPNA3 (1:5000; ab137446, Abcam, Cambridge, United Kingdom), anti-CLPP (1:2500; 15,698–1-AP, Proteintech, St. Leon-Rot, Germany), anti-GAPDH (1:5000; 0411, sc-47724, Santa Cruz Biotechnology), anti-β-actin (1:5000; AC-15, A5441, Sigma-Aldrich), anti-vinculin (1:1000; E1E9V, 13,901, Cell Signaling Technologies, Frankfurt am Main, Germany), anti-lamin A/C (1:5000; 346, sc-7293, Santa Cruz Biotechnology), anti-GST (1:2500; B-14, sc-138, Santa Cruz Biotechnology), anti-α-spectrin (1:1000; AA6, Merck), anti-GFP (1:1000; B-2, sc-9996, Santa Cruz Biotechnology), anti-LC3 (1:1000; MBL-PD014, MBL International, Woburn, MA, US), anti-p62 (1:1000; 5114, Cell Signaling Technologies), and IRDye secondary antibodies goat anti-mouse 680LT, goat anti-mouse 800CW, goat anti-rabbit 680RD, goat anti-rabbit 800CW (all 1:10,000; LI-COR Biosciences, Bad Homburg, Germany), and Alexa Fluor 555 (1:500; Thermo Fisher Scientific).
5
Brain Tissue Homogenization and Western Blotting
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Brain tissue was homogenized in an equal ratio of tissue homogenate buffer (50 mM Tris-HCl pH 7.5, 150 mM KCl, 320 mM sucrose, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and lysis buffer (1% Triton, 10% glycerol, 120 mM NaCl, 25 mM HEPES, 1 mM EDTA, 0.75 mM MgCl2, 2 mM NaF, 1 mM sodium vanadate, protease inhibitor cocktail). Alternatively, transfected HEK293T were washed in ice-cold PBS and lysed in Triton lysis buffer (1% Triton, 10% glycerol, 120 mM NaCl, 25 mM HEPES, 1 mM EDTA, 0.75 mM MgCl2, 2 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors). Samples were incubated on ice for 10 min and then centrifuged at 11,000× g for 10 min at 4 °C to separate nuclei from residual tissue. Total protein concentration was determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA), and equal amounts were loaded on SDS-PAGE, transferred to a nitrocellulose membrane, and blocked in an Odyssey blocking buffer. Membranes were incubated with primary and secondary antibodies and imaged using the Odyssey CLx imaging system (LI-COR Biosciences, Lincoln, NE, USA). Anti-FAK (clone 77; 610088) was obtained from BD Biosciences. Anti-FLAG (clone M2; F3165) and anti-β actin (clone AC-15; A5441) were obtained from Sigma-Aldrich. Secondary antibodies (goat anti mouse 680LT and goat anti rabbit 800CW) were obtained from Li-COR Biosciences.
6
Src and p300 Inhibitor Protocol
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The Src inhibitor dasatinib (ChemieTEK, Indianapolis, IN) and the p300 inhibitor C646 (Sigma Aldrich, St. Louis, MO) were purchased from the designated sources. Antibodies used were as follows: Src (B-12), p300 (C20), GAPDH (6C5), β-Tubulin (C10), Grim19, (Santa Cruz Biotechnology, Santa Cruz, CA), Src (36D10), pY-416 Src (2101), HMGA2 (D1A7), SMYD3 (D2Q4V), Lamin A/C (#2032), Calnexin (C5C9), HDAC1 (10E2), Histone H3 (D1H2), p-TYR-100 (Cell Signaling Technology, Danvers, MA), pY418 Src (92633), p300 (507) (Novus Biologicals, Littleton, CO), HMGA2 (AF3184) (R&D Systems, Minneapolis, MN), and SMYD3 (ab16027) (Abcam, Cambridge, UK). Secondary antibodies used were Donkey-anti-Rabbit 800CW and Goat-anti-Mouse 680LT (Licor, Lincoln NE).
7
Fibronectin and Histidinol Immunoblotting Protocol
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Fibronectin from human plasma (F0895) and l -histidinol dihydrochloride (H6647) were obtained from Sigma-Aldrich. Hygromycin (#ant-hg) was obtained from InvivoGen. For Western blot, anti-Pyk2 (3292) was obtained from Cell Signaling Technology; anti-FAK (610088) and anti CrkI/II (610036) were obtained from BD Transduction Laboratories; anti-CrkL (sc-319) was obtained from Santa Cruz Biotechnology; anti-GFP (11814460001) was obtained from Roche Life Science; anti–β-actin (clone AC-15) (A5441) was obtained from Sigma-Aldrich; anti–HSC-70 (1427) was obtained from Abcam. Secondary antibodies (goat anti-mouse 680LT and goat anti-rabbit 800CW) were obtained from LI-COR Biosciences. For immunofluorescence, antivinculin (V9131) was obtained from Sigma-Aldrich; rhodamine-labeled phalloidin and Alexa Fluor–conjugated secondary antibodies were obtained from Molecular Probes.
8
Regulation of B Cell Signaling Cascades
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Isolated splenocytes were starved in serum-free Iscove’s modified Dulbeco’s medium (IMDM) with 50 μM β-mercaptoethanol (Gibco, Life Technologies) for 1 hour. Subsequently, B lymphocytes were activated for 5 minutes at room temperature with H2O2 (4 mM) and anti-mouse IgM (10 μg/mL, 1022-01, Southern Biotech). Generation of whole-cell lysates and immunoblotting analysis were performed as previously.25 (link),27 (link) The antibodies used for western blotting were anti-actin (A5441, Sigma-Aldrich), anti-BTK (270-284, Sigma-Aldrich), anti-BTK pY551 (24a/BTK, BD Biosciences), anti-BTK pY223 (EP420Y, Abcam), anti-PLCG2 (Rabbit polyclonal, Biotech), and anti-PLCG2 pY753 (polyclonal, Abcam). The secondary antibodies, goat anti-mouse 800CW, goat anti-rabbit 800CW, goat anti-mouse 680LT, and goat anti-rabbit 680, were purchased from LI-COR Biosciences GmbH, Lincoln, NE, USA. Odyssey Imager from LI-COR Biosciences GmbH was used for membrane scanning, and the signals of total and phosphorylated proteins were quantified by NIH ImageJ 1.52a.
All experiments were approved by the local animal experimentation ethics committee, ID 1679. Mice were generated and maintained on C57BL/6 background. Analyzed Y223F mice and controls were sex- and age-matched. Experiments were performed on 7- and 22-week-old mice. Auto-antibody analysis was performed on 14- to 16-month-old animals.
All experiments were approved by the local animal experimentation ethics committee, ID 1679. Mice were generated and maintained on C57BL/6 background. Analyzed Y223F mice and controls were sex- and age-matched. Experiments were performed on 7- and 22-week-old mice. Auto-antibody analysis was performed on 14- to 16-month-old animals.
9
Immunoprecipitation and Western Blotting
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Immunoprecipitation analysis was carried out using Dynabeads protein G (Life Technologies) according to the manufacturer’s protocol. Cell lysates were obtained, after washing cells in PBS twice, by incubation with lysis buffer (50 mM Hepes, pH 7.0, 120 mM NaCl, 10% Glycerol, 1% NP-40, 0.5% Sodium deoxycholate) supplemented with protease inhibitors (Complete Mini, Roche) for 30 min with repeated vortexing and the lysates were cleared by centrifugation. Proteins were separated on gradient 4–12% SDS Bis-Tris NuPAGE gels (Life technologies) and transferred onto nitrocellulose membranes using the Iblot system (Life technologies). The membranes were then blocked with LI-COR Blocking Buffer (LI-COR Biosciences GmbH) and probed with specific primary antibodies. The membranes were scanned with the Odyssey infrared imaging system (LI-COR Biosciences GmbH). The densities of target bands were quantified using the application software of the Odyssey infrared imaging system. The following secondary antibodies: goat anti-mouse-800CW, goat anti-rabbit-800CW, goat anti-mouse-680LT, or goat anti-rabbit-680 (LI-COR Biosciences GmbH) were used at 1:20,000 dilutions for 1 h at room temperature.
10
Western Blot Analysis of Desmoglein and Desmoplakin
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Cells were homogenized in radioimmunoprecipitation assay buffer containing SDS, NP-40 (EMD Millipore), and protease/phosphatase inhibitor (Complete, Roche). All lysates were clarified by centrifugation. Protein concentrations were measured by the bicinchoninic acid method (Pierce, Thermo Fisher Scientific). A quantity of 65 µg per sample was loaded in 8% SDS-polyacrylamide gels under denaturing conditions. The size-fractionated proteins were electroblotted to polyvinylidene difluoride (Bio-Rad) membranes. The membranes were treated with Intercept Blocking Buffer (LI-COR) for 1 h and incubated overnight with appropriate primary antibodies sequentially in Intercept Antibody Diluent (LI-COR). Membranes were washed and incubated with secondary antibodies (goat anti-rabbit RDye 800 CW and goat anti-mouse 680 LT; LI-COR) for 1 h and washed with TBS with Tween. Images were captured using the Odyssey CLx Imaging system (LI-COR). Primary antibodies were Dsg3 (clone 5G11, Thermo Fisher Scientific), DP I+II (ab127941, Abcam), DP 1&2 (CBL173, Millipore), DP pS2849 (600-401-J65, Rockland), Vinculin (V9131, Millipore Sigma), β-tubulin (2146, Cell Signaling Technology), GFP (ab290, Abcam), and mCherry (ab167453, Abcam).
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