Protein extracts were separated using 10% SDS-PAGE gels and proteins were transferred onto PVDF membrane (Millipore) for blotting. Antibodies used for blotting: anti-HA-tag (1:2000, Santa Cruz Biotechnology, sc-7392), anti-GFP (1:1000, Chromotek, 3H9), IRDye 800CW goat anti-Mouse (1:2000, LI-COR, 926-32210), IRDye 800CW goat anti-Rat (1:2000, LI-COR, 926-32219), and IRDye 800CW Streptavidin (1:2000, LI-COR, 926-32230). Membrane blotting and washing were performed according to the manufacturer’s instructions (LI-COR). After blotting, the membranes were visualized using the LI-COR Odyssey 9120 imaging system.
Irdye 800cw goat anti rat
Manufactured by LI COR
Sourced in United States
The IRDye 800CW goat anti-Rat is a near-infrared fluorescent secondary antibody. It is designed for detection and quantification of rat proteins in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw goat anti rabbit, by LI COR (4 mentions)
Irdye 800cw goat anti mouse, by LI COR (3 mentions)
Odyssey system, by LI COR (3 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Hif2a nb100 132b, by Novus Biologicals (2 mentions)
Baf645, by R&D Systems (2 mentions)
Irdye 680rd goat anti rabbit, by LI COR (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Odyssey 9120, by LI COR (1 mentions)
Anti gfp, by Proteintech (1 mentions)
Irdye 800cw streptavidin, by LI COR (1 mentions)
Anti ha tag, by Santa Cruz Biotechnology (1 mentions)
Hif1a, by Cayman Chemical (1 mentions)
Odyssey fc, by LI COR (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Image studio 4, by LI COR (1 mentions)
Rabbit anti cblb, by Cell Signaling Technology (1 mentions)
10 protocols using irdye 800cw goat anti rat
1
Western Blot Analysis of Tagged Proteins
2
Western Blot Analysis of Ear Tissue
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Example 4
Western Analysis.
Ear tissue samples (3 ear hole donuts/ear from 3 separate mice) were homogenized in radio-immunoprecipitation assay buffer (50 mM Tris-HCl pH 7.6, containing 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA and 0.1% SDS) with 1 mM PMSF and a protease inhibitor cocktail (Sigma). Samples with equal amounts of protein (about 40 μg) were loaded into a NuPAGE 4-12% Bis-Tris gradient gel or 8% Bis-Tris gel (Life Technologies, Grand Island, N.Y.), electrophoresed and then electro-transferred onto a PVDF-FL membrane (Immobilon, Billerica, Mass.). The membrane was subsequently blocked with Odyssey blocking buffer (LI-COR, Lincoln, Nebr.), probed with primary antibodies (HIF1a (Ser. No. 10/006,421, Cayman Chemical, Ann Arbor, Mich.), HIF2a (NB100-132B, Novus, Littleton, Colo.), Wnt5a (BAF645, R&D System) or a-Tubulin (Sigma) overnight at 4° C., then further incubated with Alexa Fluor-labeled secondary antibodies (IRDye 800CW goat-anti rat or IRDye 800CW goat-anti rabbit (LI-COR, Lincoln, Nebr.) for 1 hr and scanned using the Odyssey system (LI-COR, Lincoln, Nebr.).
3
Western Blotting Analysis of Ear Tissue
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Example 4
Western Analysis.
Ear tissue samples (3 ear hole donuts/ear from 3 separate mice) were homogenized in radio-immunoprecipitation assay buffer (50 mM Tris-HCl pH 7.6, containing 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA and 0.1% SDS) with 1 mM PMSF and a protease inhibitor cocktail (Sigma). Samples with equal amounts of protein (about 40 μg) were loaded into a NuPAGE 4-12% Bis-Tris gradient gel or 8% Bis-Tris gel (Life Technologies, Grand Island, N.Y.), electrophoresed and then electro-transferred onto a PVDF-FL membrane (Immobilon, Billerica, Mass.). The membrane was subsequently blocked with Odyssey blocking buffer (LI-COR, Lincoln, Nebr.), probed with primary antibodies (HIF1a (10006421, Cayman Chemical, Ann Arbor, Mich.), HIF2a (NB100-132B, Novus, Littleton, Colo.), Wnt5a (BAF645, R&D System) or a-Tubulin (Sigma) overnight at 4° C., then further incubated with Alexa Fluor-labeled secondary antibodies (IRDye 800CW goat-anti rat or IRDye 800CW goat-anti rabbit (LI-COR, Lincoln, Nebr.) for 1 hr and scanned using the Odyssey system (LI-COR, Lincoln, Nebr.).
4
CBLB Protein Quantification in Cells
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Three to five million cells were resuspended in RIPA Lysis and Extraction Buffer (ThermoFisher, Scientific, Waltham, MA, USA) supplemented with 1X Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher, Scientific, Waltham, MA, USA) and incubated on ice for 20 min. The soluble fraction was recovered after 10 min of centrifugation at 10,000× g 4 °C. Protein concentration was determined by standard Bradford assay. A total of 20 µg of protein was loaded in a Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA, USA) and separated by electrophoresis. The gel was transferred to a Midi format 0.2 µm PVDF membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membrane was blocked with EveryBlot Blocking Buffer (Bio-Rad, Hercules, CA, USA) and incubated for 1 h at room temperature (RT) or overnight at 4 °C with rabbit anti-CBLB (Cell Signaling Technologies, Danvers, MA, USA, clone: D3C12) diluted at 1:500 and rat anti-GAPDH (Biolegend, San Diego, CA, USA, clone: W17079A) diluted at 1:1000 in blocking buffer. After washing, the membrane was incubated for 1 h at room temperature with IRDye 800CW goat anti-rat and IRDye 680RD goat anti-rabbit (LI-COR Biosciences, Lincoln, NE, USA) at 1:15,000 in a blocking buffer. The membrane was developed using LI-COR Odyssey Fc and band intensity was quantified with Image Studio 4.0 (LI-COR Biosciences, Lincoln, NE, USA).
5
Yeast Protein Extraction and Immunoblotting
6
Quantifying Phosphorylation Dynamics in HEK293T
7
Western Blot Analysis of Apoptosis Markers
8
Western Blot Analysis of HA and Actin
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Cells were lysed using either 1.5x Laemilli buffer at a concentration of 10,000 cells/μL or standard RIPA buffer supplemented by 23.4 μM Leupeptin (Roche), 6.1 μM Aproptinin (Roche), 14.5 μM Pepstatin A (Roche), 0.1 mM PMSF (Millipore), and 1 mM Sodium Orthandovate. Samples were then boiled at 100°C for 10 minutes. Extracts were loaded at a range of 50,000–150,000 cells or 5–15 μg of protein and separated on 12% acrylamide gels under denaturing and reducing conditions. Gels were transferred onto 0.45 μm nitrocellulose membranes. Blots were developed using LI-COR's Odyssey imaging system. The following primary antibodies were used: rat anti-HA (Roche) at 1:1000 and rat anti-actin (Abcam) at 1:10,000. Secondary antibodies were as follows: goat anti-rat IRDye 800CW (Li-Cor Biosciences) and goat anti-rabbit IRDye 680RD (Li-Cor Biosciences).
9
Western Blot Analysis of CD14 Expression
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The tissues or cells were lysed in radio-immunoprecipitation lysis buffer (RIPA) with protease inhibitors cocktails (Roche, 04693116001). Following centrifugation at 12,000 g for 15 min at 4°C, the supernatants were collected. Western blot was conducted as described previously. Briefly, 10% SDS-PAGE gels were used to separate protein samples. After proteins transferred to polyvinylidene fluoride membranes, the membranes were blocked with 5% non-fat milk in Tris saline buffer containing 0.1% Tween-20 (TBST) for 1 h at room temperature. Rat anti-CD14 antibody (BD pharmingen, 557896, 1:1,000), and mouse anti-β-actin antibody (Proteintech, 66009-1-Ig, 1:10,000) were used to detect CD14 and β-actin overnight at 4°C. Goat anti-rat IRDye 800CW (LI-COR Biosciences, 926-32219) or goat anti-mouse IRDye 800CW (LI-COR Biosciences, 926-32210) were used to detect CD14 and actin, respectively. LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Nebraska) was used for CD14 and actin detection.
10
Immunoprecipitation and Western Blotting of Parasite Proteins
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For Western blotting analysis, the samples were lysed in 300 μL IP buffer and incubated with 30 μL Pierce anti-HA magnetic beads (Thermo) overnight at 4°C, following which the beads were washed three times with IP buffer and the bound proteins eluted with 30 μL 3x Sample Loading Buffer (NEB) at 95°C for 10 min. 5% of the post-IP lysate supernatant and 15% of the immunoprecipitate were separated by SDS-PAGE and transferred to a nitrocellulose membrane as above. The membrane was blocked with 2% w/v skim milk powder, 0.1% v/v Tween 20 in PBS for 1 h at room temperature, then incubated with primary antibodies in blocking buffer overnight at 4°C. Primary antibodies used were: 1:1000 rat anti-HA (Roche #11867423001), 1:1000 mouse anti-ROP1 (Abnova #MAB17504), 1:1000 rabbit anti-C1QBP (Abcam #ab270032), 1:10,000 rabbit anti-GAPDH (Proteintech #10494-1-AP), and 1:1000 rabbit anti-GRA29 [15 (link)]. Blots were stained with secondary antibodies for 1 h at room temperature: 1:10,000 goat anti-mouse IRDye 680LT (Li-Cor #925–68020), 1:10,000 goat anti-rat IRDye 800CW (Li-Cor #925–32219), 1:10,000 donkey anti-rabbit IRDye 680LT (Li-Cor #925–68023), and 1:10,000 donkey anti-rabbit IRDye 800CW (Li-Cor #925–32213). Blots were visualised using an Odyssey CLx scanner (Li-Cor).
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