Total cell lysates were prepared in standard RIPA extraction buffer containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, Missouri). Thirty micrograms of protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL). The membranes were immunoprobed with FAK, Phospho-FAK (Y397) (Cell Signaling, Danvers, MA) or Actin (C-2) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight separately. Next, membranes were incubated with IRDye 800-conjugated goat anti-mouse IgG (Rockland, Immunochemicals, Gibertsville, PA) or Alexa Fluor 680 goat anti-rabbit IgG (Life Technologies, Grand Island, NY) as secondary antibodies. Bands were detected using Li-Cor Odyssey Imaging System (Li-Cor, Lincoln, NE).
Irdye 800 conjugated goat anti mouse igg
Manufactured by Rockland Immunochemicals
Sourced in United States
IRDye 800-conjugated goat anti-mouse IgG is a secondary antibody reagent used for detection and visualization in various immunoassay and imaging applications. It is conjugated with the IRDye 800 fluorescent dye, which allows for near-infrared signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 680 goat anti rabbit igg, by Thermo Fisher Scientific (4 mentions)
Odyssey imaging system, by LI COR (4 mentions)
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Phospho fak y397, by Cell Signaling Technology (1 mentions)
Actin c 2, by Santa Cruz Biotechnology (1 mentions)
Tunel assay kit, by Promega (1 mentions)
Triphenyl tetrazolium chloride (ttc), by Merck Group (1 mentions)
Mouse monoclonal antibody against cox 4, by Abcam (1 mentions)
Rabbit polyclonal antibody against caspase 3, by Abcam (1 mentions)
Donkey anti rabbit igg, by Rockland Immunochemicals (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Mouse monoclonal anti gfp antibody, by Roche (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti survivin, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
6 protocols using irdye 800 conjugated goat anti mouse igg
1
Western Blot Analysis of FAK Signaling
2
Apoptosis Pathway Analysis Protocols
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TUNEL assay kit was purchased from Promega (Madison, WI). MTT, TTC, JC-1, DCFH-DA, Hoechst 33342, rabbit polyclonal antibody against Bax, Bcl-2, Bcl-xL, AIF, or histone H1, mouse monoclonal antibody against β-Tubulin III, or β-actin were purchased from Sigma (St. Louis, MO). Mouse monoclonal antibody against Cytochrome C was purchased from BD Pharmingen (San Diego, CA). Rabbit polyclonal antibody against cleaved caspase-3 (Asp175) was purchased from Cell signaling Technology (Danvers, MA). Rabbit polyclonal antibody against caspase-3 and mouse monoclonal antibody against COX IV was purchased from Abcam (Cambridge, Mass., UK). IRDye 800 conjugated goat anti-mouse IgG or donkey anti-rabbit IgG was purchased from Rockland (Gilbertsville, PA). Mammalian protein extraction reagent (M-PER), tissue protein extraction reagent (T-PER), nuclear and cytoplasmic extraction reagents (NE-PER), mitochondria isolation kit and Coomassie plus Bradford assay kit were purchased from Thermo Scientific Pierce (Rockford, IL). All standard culture reagents were obtained from Gibco (Grand Island, NY).
3
Western Blot Analysis of Cellular Proteins
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For Western blot analysis, 1–2 × 107 cells were collected from log phase cultures for total protein extraction by TCA precipitation as described previously (Keaton et al., 2008 (link)). Electrophoresis and Western blotting were performed as described previously (Bose et al., 2001 (link)). Monoclonal mouse anti-GFP antibodies (Roche) were used at 1:500 dilution. Rabbit polyclonal anti-Cdc11 antibodies (Santa Cruz Biotechnology, Inc.) were used at 1:5,000 dilution. Fluorophore-conjugated secondary antibodies against mouse (IRDye800 conjugated goat anti–mouse IgG; Rockland Immunochemicals) or antibodies against rabbit (Alexa Fluor 680 goat anti–rabbit IgG, Invitrogen) were used at 1:5,000 dilution. Blots were visualized by the ODYSSEY imaging system (LI-COR Biosciences).
4
Western Blot Analysis of Cell Signaling
5
TCA-Based Protein Extraction and Western Blotting
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Samples were prepared using TCA (Kozubowski et al., 2008 (link)) from ∼1 × 107 cells. Cells were collected and resuspended in pronase buffer (25 mM Tris-HCl, pH 7.5, 1.4 M Sorbitol, 20 mM NaN3, and 2 mM MgCl2). TCA (Sigma-Aldrich) was added to a final concentration of 17% wt/vol, and pellets were stored at −80°C. Cells were thawed and homogenized by vortexing with glass beads at 4°C for 10 min. Precipitated proteins were collected from lysates by centrifugation at 4°C and resuspended in sample buffer (40 mM Tris-HCl, pH 6.8, 8 M Urea, 5% SDS, 143 mM β-mercaptoethanol, 0.1 mM EDTA, and 0.4 mg/ml bromophenol blue), and the pH was titrated using 2 M Tris-HCl, pH 8.0. After SDS-PAGE and transfer, blots were probed using monoclonal mouse anti-GFP and anti-HA antibodies (Roche) at 1:1,000 dilution and anti-Cdc11 antibody (Santa Cruz Biotechnology) at 1:10,000 dilution. Fluorophore-conjugated secondary antibodies for mouse (IRDye800-conjugated goat anti–mouse IgG; Rockland Immunochemicals) or rabbit (Alexa Fluor 680 goat anti–rabbit IgG; Invitrogen) were used at 1:5,000 dilution. Western blots were visualized using the ODYSSEY imaging system (LI-COR Biosciences).
6
Western Blot Analysis of IκBα
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After stimulation, ECs were lysed in lysis buffer (25 mM Tris pH 7.5, NaCl 150 mM, Triton X-100 1%, Sodium Deoxycholate 1%, SDS 0,1%, 2 mM EDTA, 1% (v/v) Nonidet P-40, and 1mM DTT) containing inhibitors of proteases (Sigma-Aldrich), and phosphatases (Sigma-Aldrich), and following a 15-min incubation on ice, cell debris were spun down (12,000 × g, 20 min, 4 °C) and the supernatants were frozen and stored at −80 °C. Small aliquots of the various extracts were routinely processed for protein content determination, by using a protein assay kit (Bio-Rad, Milan,Italy). For Western blot analysis, cell extracts were subjected to immunoblots by described procedures23 (link). Detection was carried out with Alexa FluorR 680-conjugated goat anti-rabbit Abs (Invitrogen) and IRDye™ 800-conjugated goat anti-mouse IgG (Rockland, Gilbertsville, PA, USA) secondary Abs. Blotted proteins were detected and quantified using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Anti-IκBa (sc-371) was obtained from Santa Cruz Biotechnology while anti-β-tubulin (T5293) was from Sigma-Aldrich.
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