Tissues were thawed and homogenized in radioimmunoprecipitation buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 0.1% sodium deoxycholate, and 1 mM EDTA) containing PhosSTOP phosphatase inhibitor (Roche #04906845001) and complete Mini protease inhibitor (Roche #11836153001) using a Precellys 24 high-throughput homogenizer (Bertin Technologies, Rockville, Washington D.C., USA). Protein content was measured by the Bradford method, and samples were normalized. 20 mg of total protein was separated by SDS-PAGE on polyacrylamide gels (15%) and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc.). The membrane was probed with primary antibodies listed in the antibody section. Anti-Actin, anti-Gapdh and anti-Hprt antibodies were used as loading controls. Proteins were detected with secondary antibodies conjugated to horseradish peroxidase (RPN4201 and RPN4301) and the ECL detection kit (both GE Healthcare) or by near-infrared fluorescent dyes (LI-COR). Visualization of protein bands was realized by LAS-4000 mini (Fujifilm) or with an Odyssey near-infrared scanner (Licor Biosciences). Band intensities were measured by using ImageJ (National Institutes of Health).
Odyssey near infrared scanner
Manufactured by LI COR
Sourced in United States
The Odyssey near-infrared scanner is a laboratory instrument designed for the detection and quantification of fluorescent and infrared signals in a variety of applications. It utilizes near-infrared fluorescence technology to provide sensitive and accurate measurements. The core function of the Odyssey scanner is to enable the visualization and analysis of target proteins, nucleic acids, and other biomolecules in a range of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Mouse monoclonal anti α tubulin, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
α mouse irdye680rd, by LI COR (2 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (2 mentions)
Skimmed milk powder, by Merck Group (2 mentions)
Las 4000 mini, by Fujifilm (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Near infrared fluorescent dyes, by LI COR (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
Las 4000, by Cytiva (1 mentions)
Anti rabbit irdye 800cw, by LI COR (1 mentions)
Rabbit monoclonal anti gapdh, by Cell Signaling Technology (1 mentions)
Bio plex cell lysis kit, by Bio-Rad (1 mentions)
Revert total protein stain, by LI COR (1 mentions)
Image studio version 5, by LI COR (1 mentions)
Total protein stain, by LI COR (1 mentions)
Rabbit anti myc, by Cell Signaling Technology (1 mentions)
23 protocols using odyssey near infrared scanner
1
Protein Extraction and Quantification
2
Western Blot Analysis of Centromere Proteins
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Whole-cell extracts were resolved by SDS-PAGE and blotted onto Nitrocellulose membranes. Membranes were blocked in TBS-Tween (10% powdered milk) or Odyssey blocking buffer (Li-cor Biosciences) and incubated overnight at 4°C with the indicated antibodies. Secondary antibodies were used at 1 : 10 000 prior to detection on Odyssey near-infrared scanner (Li-cor Biosciences).
The following primary antibodies were used for immunoblot: rabbit polyclonal anti-CENP-A (no. 2186, Cell Signaling Technology) at 1 : 500, rabbit polyclonal anti-CENP-B (no. ab25734, Abcam) at 1 : 200, rabbit polyclonal anti-CENP-T (no. ab220280, Abcam) at 1 : 250, guinea pig polyclonal anti-CENP-C (no. PD030, MBL International) at 1 : 250, rabbit polyclonal anti-H4K20me (no. ab9052, Abcam) at 1 : 4000, rabbit anti-CENP-E (kind gift from Don Cleveland) 1 : 250, mouse monoclonal anti-Hec1 (no. MA1-23308, Thermo Fischer Scientific) at 1 : 250, mouse monoclonal anti-α tubulin (T9026, Sigma-Aldrich) at 1 : 5000 and rabbit monoclonal anti-GAPDH (no. 2118S, Cell Signaling) at 1 : 2000. Secondary antibodies used: IRDye800CW anti-rabbit (Li-cor Biosciences), IRDyLight800CW anti-rabbit (Li-cor Biosciences), IRDyLight800CW anti-guinea (Li-cor Biosciences), IRDyLight800CW anti-mouse (Li-cor Biosciences) and IRDyLight680LT anti-mouse (Li-cor Biosciences).
The following primary antibodies were used for immunoblot: rabbit polyclonal anti-CENP-A (no. 2186, Cell Signaling Technology) at 1 : 500, rabbit polyclonal anti-CENP-B (no. ab25734, Abcam) at 1 : 200, rabbit polyclonal anti-CENP-T (no. ab220280, Abcam) at 1 : 250, guinea pig polyclonal anti-CENP-C (no. PD030, MBL International) at 1 : 250, rabbit polyclonal anti-H4K20me (no. ab9052, Abcam) at 1 : 4000, rabbit anti-CENP-E (kind gift from Don Cleveland) 1 : 250, mouse monoclonal anti-Hec1 (no. MA1-23308, Thermo Fischer Scientific) at 1 : 250, mouse monoclonal anti-α tubulin (T9026, Sigma-Aldrich) at 1 : 5000 and rabbit monoclonal anti-GAPDH (no. 2118S, Cell Signaling) at 1 : 2000. Secondary antibodies used: IRDye800CW anti-rabbit (Li-cor Biosciences), IRDyLight800CW anti-rabbit (Li-cor Biosciences), IRDyLight800CW anti-guinea (Li-cor Biosciences), IRDyLight800CW anti-mouse (Li-cor Biosciences) and IRDyLight680LT anti-mouse (Li-cor Biosciences).
3
Quantifying PLIN5 Protein Levels in Human Muscle
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Protein levels of PLIN5 in human muscle tissue were determined by Western blot in 8/10 individuals from the subgroup. Tissues were lysed using Bio‐Plex Cell Lysis kit (171‐304011; Bio‐Rad) and equal amounts of protein were loaded onto the gels (Figure S1 ). After gel electrophoresis, proteins were transferred by Western blotting and a Revert total protein stain (LI‐COR Bioscience, Westburg) was performed to determine the protein quality and the protein quantity on the blots. Then, the membranes were blocked with LI‐COR Blocking buffer for 30 min and incubated with a primary antibody against PLIN5 (1:2500, diluted in LI‐COR Blocking buffer) overnight at room temperature. A IRDye800‐conjugated secondary antibody was used for visualization PLIN5 by an Odyssey near infrared scanner (LI‐COR Biosciences). Western blots were quantified with Image Studio version 5.2 (LI‐COR Biosciences) and values were normalized to total protein stain.
4
Quantifying Intestinal Albumin Leakage
5
Immunoblotting of RHEB, TSC1/2, and pS6K
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After transfection, HEK293T cells were transferred on ice, washed with PBS (4°C), and lysed in 70 μl 50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 50 mM NaF, 1% Triton X100 in the presence of protease and phosphatase inhibitors (Complete, Roche Molecular Biochemicals, Woerden, the Netherlands). Cell lysates were subjected to immunoblotting using the following primary antibodies: anti-RHEB mouse monoclonal [62 (link)], anti-TSC1 and TSC2 rabbit polyclonal [63 (link)], T389-phosphorylated S6K (1A5, #9206, Cell Signaling Technology), and rabbit anti-myc (#2272, Cell Signaling Technology), all 1:1,000. Primary antibody binding was assessed by incubation with goat anti-rabbit (680 nm) and anti-mouse (800 nm) conjugates (1:15,000, Li-Cor Biosciences, Lincoln, USA) followed by detection on an Odyssey near-infrared scanner (Li-Cor Biosciences).
6
Immunoblotting for Akt Signaling Proteins
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Samples were lysed in RIPA buffer containing inhibitors and heated at 95°C for 5 min before electrophoresis. Proteins were transferred to a 0.2-mm nitrocellulose membrane, blocked with 5% nonfat dry milk, and incubated with primary antibodies at 4°C overnight. Immunoblotting was conducted with the following antibodies: IPMK from Covance, p-Akt-S473, p-Akt-T308, t-Akt, and GAPDH from Cell Signaling Technology. Blots were imaged and quantitated using an Odyssey Near-Infrared Scanner (Li-Cor Biosciences, Lincoln, NE, USA).
7
Two-Dimensional Protein Separation
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Directly after IEF, proteins were reduced in sample buffer (150 mM Tris-HCl, pH 8.5, 2% (w/v) lithium dodecyl sulfate (LDS), 10% (v/v) glycerol, 0.51 mM EDTA, 0.22 mM Serva Blue G250, 0.175 mM Phenol Red) containing 50 mM DTT and subsequently alkylated in sample buffer containing 125 mM iodoacetamide (IAA) for 15 min each. For protein separation in the second dimension, IPG strips were placed on precast NuPAGE Novex 4%–12% Bis-Tris ZOOM Protein Gels, 1.0 mm, IPG-well (Thermo Fisher Scientific, Waltham, MA USA), 4 µL Dual Color Protein Standard III (Serva) was used as molecular weight marker and electrophoresis was performed in MES buffer at 180 V. Gels were fixed in 40% (v/v) ethanol, 10% (v/v) acetic acid for 60 min, rinsed 3× for 10 min in water and stained with colloidal Coomassie staining solution (0.08% (w/v) Coomassie Brilliant Blue G-250, 1.6% (w/v) ortho-phosphoric acid, 8% (w/v) ammonium sulfate and 10% (v/v) methanol) over night. Excess dye was removed by washing in water. Gels were imaged using an Odyssey near-infrared scanner (LI-COR, Lincoln, NE, USA) at 700 nm wave length, intensity set to 5.0, quality set to “high” and resolution to 84 µm.
8
Optimized Western Blot Transfer Protocol
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Gels stained with colloidal Coomassie were equilibrated twice in MES buffer for 10 min. Proteins were transferred to PVDF membrane (Millipore, Darmstadt, Germany) in transfer buffer (25 mM Bicine, 25 mM Bis-Tris, 1.025 mM EDTA, 10% (v/v) methanol) in a XCell II Blot Module (Thermo Fisher Scientific) at 30 V for 13 min. Transfer time was empirically optimized for myelin proteins and may need adaptation for other samples and other blotting equipment. After transfer, gels were re-stained with colloidal Coomassie and imaged as described above. All edges of the PVDF membranes were labeled with cross-shaped orientation marks visible in both near-infrared channels to facilitate later overlay of membrane images obtained after colloidal Coomassie staining (700 nm channel) and immunodetection (800 nm channel, see below). A conventional blue laboratory pen (e.g., Lumocolor Permanent, Staedtler, Nürnberg, Germany) served this purpose. Membranes were imaged using an Odyssey near-infrared scanner (LI-COR) at 700 nm wave length, intensity set to 2.0, quality set to “high” and resolution to 84 µm.
9
Cardiac Protein Expression Analysis
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Protein analyses from heart samples were performed as described previously [15 (link)]. In brief, whole hearts were homogenized, proteins were separated by SDS-PAGE on polyacrylamide gels, and transferred onto Protran nitrocellulose membranes (GE Healthcare). Membranes were incubated with primary antibodies against AKT1 (#2967), AKT2 (#3063), GSK3β (#9315), and GAPDH (#2118) from Cell Signaling Technology, or p38 (ab170099) from Abcam. Secondary antibodies used were α-rabbit or α-mouse IRDye800CW and α-rabbit or α-mouse IRDye680RD from LI-COR Biosciences. Signals were detected and quantified with an Odyssey near-infrared scanner (LI-COR Biosciences).
10
Binding Affinity Determination of Nanobodies
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Purified rat PDGFRβ (Sino-Biological, Eschborn, Germany) was coated overnight at 4 °C at 1 μg/mL in PBS in ELISA plates (Nunc MaxiSorp™, Thermo Fisher Scientific, Bleiswijk, The Netherlands). Next day, plates were washed with PBS and blocked with PBS, supplemented with 4% (w/v) skimmed milk powder (Merck Millipore, Darmstadt, Germany) and 0.05% v/v Tween, for 1 h at RT. All further incubations were carried out for 1 h at RT in PBS supplemented with v/v 0.05% Tween (PBST). A fixed concentration of IRDye800 conjugated sdAb was mixed with unconjugated competitors (sdAbs or ligand) in three-fold serial dilutions, starting with a 20-fold molar excess. Fluorescent signal of the IRDye800 conjugated sdAb was detected using the Odyssey near-infrared scanner (LI-COR Biosciences, Lincoln, NE, USA).
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