Protein concentrations were determined using the BCA Protein Assay kit (ThermoFisher Scientific). Western blotting was then performed as described previously [32 (link)]. Primary antibodies against pThr (5267) and β-Actin (1616) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Nur77 antibody was from Abcam (109180; Cambridge, MA). The secondary antibodies, donkey anti-goat IRDye 680RD (926–68074) and goat anti-rabbit IRDye 800CW (925–32211), were obtained from LI-COR Biosciences (Lincoln, NE). The infrared signal was detected with an Odyssey Infrared Imager (LI-COR Biosciences). The densitometric analysis was performed using Image Studio software (LI-COR Biosciences).
Irdye 680rd donkey anti goat
Manufactured by LI COR
Sourced in United Kingdom, United States
The IRDye 680RD Donkey anti-Goat is a secondary antibody labeled with the IRDye 680RD fluorescent dye. It is designed to detect and bind to goat primary antibodies for use in various immunoassay and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 680rd donkey anti rabbit, by LI COR (4 mentions)
Irdye 800cw goat anti rabbit, by LI COR (3 mentions)
Irdye 800cw donkey anti mouse, by LI COR (3 mentions)
Odyssey infrared imager, by LI COR (2 mentions)
Irdye 800cw goat anti mouse, by LI COR (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Image studio software, by LI COR (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti nur77 antibody, by Abcam (1 mentions)
Super signal femto, by Thermo Fisher Scientific (1 mentions)
Rabbit anti β actin a2066, by Merck Group (1 mentions)
Hif 1α antibody, by Cayman Chemical (1 mentions)
Goat anti rabbit hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse hrp, by Thermo Fisher Scientific (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Amersham ecl, by GE Healthcare (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Nb100 479, by Novus Biologicals (1 mentions)
Dl 2 amino 5 phosphonopentanoicacid sodium salt ap5, by Abcam (1 mentions)
10 protocols using irdye 680rd donkey anti goat
1
Quantifying Protein Levels by Western Blot
2
Western Blot Analysis of Protein Targets
3
Western Blot Analysis of HIF-1α and Arginine Metabolism
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Protein samples were separated on 4–12% Bis-Tris acrylamide gels (Life Technologies) and transferred to PVDF (Millipore). HIF-1α antibody was purchased from Cayman Chemical (10006421) and Novus Bio (NB100-479). Arginase I (H-52) and NOS2 (M19) antibodies were purchased from Santa Cruz Biotechnology. Rabbit anti-β-actin (A2066) was purchased from Sigma. β-tubulin (AA2) was purchased from Millipore. Secondary antibodies were goat anti-rabbit HRP (Thermo), goat anti-mouse HRP (Thermo), donkey anti-mouse IRDye800CW, donkey anti-goat IRDye680RD and donkey anti-rabbit IRDye680RD (Licor). Blots were developed with Supersignal Femto (Thermo) or Amersham ECL (GE Healthcare Life Sciences) or visualized on an Odyssey CLx imager (Licor).
4
Dynamin Phosphorylation Assay Protocol
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All general reagents were from Sigma-Aldrich (Gillingham, UK), and all tissue culture reagents were from Life Technologies (Paisley, UK) unless otherwise stated. 6-Cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX) and DL-2-amino-5-phosphonopentanoic acid sodium salt (AP5) were from Abcam (Cambridge, UK), and bafilomycin A1 was from Cayman Chemical (Ann Arbor MI, USA). Sheep anti-dynamin PSer774 and sheep anti-dynamin PSer778 were from AbD Serotec (Kidlington, UK), goat anti-dynamin I was from Santa Cruz (Dallas, USA), HRP-conjugated monoclonal mouse anti-β-actin (clone AC-15) and HRP-conjugated rabbit anti-sheep/goat were from Sigma-Aldrich. Donkey anti-goat IRDye 680RD and donkey anti-mouse IRDye 800CW were from LI-COR Biosciences (Cambridge, UK). The plasmid encoding synaptophysin-pHluorin (syp-pHluorin) was a gift from Prof. L. Lagnado (University of Sussex, UK).
5
Western Blot Analysis of Protein Interactions
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Cells were lysed 48 hours after transfection using m-PER (Thermo Scientific, Waltham, Massachusetts, USA) and 1X protease inhibitors (Roche, Basel, Switzerland). Cell lysate preparation, protein quantification and WB analysis were performed as described before (19 (link)). The following antibodies were used: anti-HA (Cell Signaling Technology, Danvers, Massachusetts, USA), anti-Myc (Cell Signaling Technology, Danvers, Massachusetts, USA), anti-FLNA (Abcam, Cambridge, UK), anti-GAPDH (Millipore, Burlington, Massachusetts, USA) and anti-actin (Santa Cruz Biotechnologies, Dallas, Texas, USA). Secondary antibodies used were IRDye 800CW Goat anti-mouse, IRDye 680RD Goat anti-Rabbit and IRDye 680RD Donkey anti-Goat (Li-Cor, Lincoln, Nebraska, USA).
6
Immunoblotting Analysis of Apoptosis Markers in Jurkat Cells
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Low-passage Jurkat T cells were seeded on a 12-well plate (100 000 cells per well per 0.5 mL). Next, staurosporine (STS, 5 μM final concentration) was added to the cells and incubated for various times (0 min, 30 min, and 1, 2, 4, 6, and 8 hours). Next, cells were harvested, centrifuged at 500×g for 5 min, washed with 1× PBS, and centrifuged again at 500×g for 5 min. The cell pellet was then sonicated and centrifuged at 14 000×g for 5 min. Then, PBS was aspirated, and cell pellets were suspended in 50 μL of 1× SDS/DTT and boiled for 5 min. Samples (20 μL) were subjected to SDS–PAGE (30 min, 200 V, 4–12% Bis-Tris 15-well gels), followed by transfer to a nitrocellulose membrane (60 min, 10 V) and blocking with 2% BSA in TBS-T (60 min, RT). Proteins were detected by probing the membrane with primary antibodies (anti-caspase 3 Cell Signaling #9662 1 : 1000; anti-PARP Cell Signaling #9542 1 : 1000; anti-caspase 6 Cell Signaling #9762 1 : 1000; anti-lamin A/C Cell Signaling #4777 1 : 1000; overnight, +4 °C) followed by incubation with fluorescently labeled secondary antibodies (IRDye 800CW donkey anti-goat, LI-COR; IRDye 680RD donkey anti-goat, LI-COR; 30 min, RT). The membranes were finally scanned in a LI-COR system (700 nm/red and 800 nm/green channels, Odyssey® CLx, Lincoln, NE, USA). The protein bands were quantified using Image Studio Lite software (Lincoln, NE, USA).
7
SDS-PAGE Analysis of Deglycosylated Proteins
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All samples were suspended in SDS sample buffer, treated with 1000 U of Endoglycosidase Hf or Endo H (respectively, for molecular weight proteins ~10-50 kDa or ~50–100 kDa; New England Biolabs, P0703S or P0702S) where indicated, and denatured for 1 h at 37 °C prior to resolution by SDS-PAGE (16% PAGE, 120 V, 120 min). To analyse the translation products, gels were fixed for 5 min (20% (v/v) MeOH, 10% (v/v) AcOH), dried for 2 h at 65 °C and the radiolabelled species visualised using a Typhoon FLA-700 (GE Healthcare) following exposure to a phosphorimaging plate for 24–72 h. Knock-down efficiencies (EMC2, EMC5, Sec61α, SRα) and controls (EMC6, OST, LMNB1, hSnd2, CAML) were determined by quantitative immunoblotting. Following transfer to a PVDF membrane in transfer buffer (0.06 M Tris, 0.60 M glycine, 20% MeOH) at 300 mA for 2.5 h, PVDF membranes were incubated in 1X Casein blocking buffer (10X stock from Sigma-Aldrich, B6429) made up in TBS, incubated with appropriate primary antibodies (1:500 or 1:1000 dilution) and processed for fluorescence-based detection as described by LI-COR Biosciences using appropriate secondary antibodies (IRDye 680RD Donkey anti-Goat, IRDye 680RD Donkey anti-Rabbit, IRDye 800CW Donkey anti-Guinea pig, IRDye 800CW Donkey anti-Mouse) at 1:10,000 dilution. Signals were visualised using an Odyssey CLx Imaging System (LI-COR Biosciences).
8
Western Blot Analysis of siRNA-Treated HeLa Cells
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Following semi-permeabilisation, aliquots of siRNA-treated HeLa cells were suspended in SDS sample buffer, denatured for 12 h at 37°C and sonicated prior to resolution by SDS-PAGE (16% or 10% PAGE, 120V, 120–150 min). Following transfer to a PVDF membrane in transfer buffer (0.06 M Tris, 0.60 M glycine, 20% MeOH) at 300 mA for 2.5 h, PVDF membranes were incubated in 1X Casein blocking buffer (10X stock from Sigma-Aldrich, B6429) made up in TBS, incubated with appropriate primary antibodies (1:500 or 1:1000 dilution) and processed for fluorescence-based detection as described by LI-COR Biosciences using appropriate secondary antibodies (IRDye 680RD Donkey anti-Goat, IRDye 680RD Donkey anti-Rabbit, IRDye 800CW Donkey anti-Mouse) at 1:10,000 dilution. Signals were visualised using an Odyssey CLx Imaging System (LI-COR Biosciences).
9
Western Blot Analysis of Mouse Muscle Proteins
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Proteins were extracted from mice brachial biceps using RIPA buffer (Life technologies) and a protease inhibitor cocktail (Life technologies). Then, 40 µg of protein was loaded on each lane and separated by SDS-PAGE on 4–12% NuPAGE Bis-Tris gels (Life Technologies), using Chameleon Duo as a size marker, and transferred onto nitrocellulose membranes (at 100 V for 3 h at 4 °C). Membranes were blocked using fluorescent WB blocking buffer (Intercept blocking buffer, Li-Cor, ref 927-60001) in TBS 1× for 1 h at room temperature. Primary antibodies (Romeo (N-terminal epitope), 1:1000, abcam 124684) were then diluted in blocking buffer and incubated overnight at 4 °C. After washing in TBS-T, membranes were then incubated with secondary antibody (IRDye 680RD Donkey anti-goat, Li-Cor, ref 926-68074), and were diluted 1:10,000 in blocking buffer for 45 min at room temperature. The GAPDH (1:1000, abcam 9483) loading control was detected using a dilution of 1:10,000 of secondary antibodies (IRDye 680RD Donkey anti-rabbit, Li-Cor, ref 926-68073). The membranes were then washed in TBS-T and revealed using NIR Fluorescence LI-COR.
10
Protein Expression Analysis by Western Blot
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