FACS-sorted splenic NK cells were lysed in a buffer containing 50 mM Tris/HCl pH 8.0, 10% (v/v) glycerol, 0.1 mM EDTA, 150 mM NaCl (all from Roth), 2 mM DTT, 0.5% NP40 (Igepal CA-630), 25 mM sodium fluoride, 0.2 mM sodium vanadate, 1 mM PMSF, 10 ng/μl leupeptin, 10 ng/μl aprotinin, 10 ng/μl pepstatin (all from Sigma-Aldrich) and lysates were cleared by centrifugation. Ten micrograms protein per lane were loaded and separated by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond, GE Healthcare). As a molecular weight standard PageRuler® Prestained Protein Ladder (Thermo Fisher Scientific) was used. Membranes were probed with anti-STAT1 (Cell Signaling Technology), anti-panERK (BD Transduction Laboratories) and IRDye fluorophor-conjugated secondary antibodies (LI-COR Biosciences). Blots were scanned with Odyssey® Classic infrared imager (LI-COR Biosciences).
Odyssey classic infrared imager
Manufactured by LI COR
The Odyssey Classic Infrared Imager is a laboratory instrument designed for high-performance infrared imaging. It utilizes near-infrared fluorescence detection technology to enable quantitative analysis of a variety of samples, including proteins, nucleic acids, and small molecules. The Odyssey Classic provides high sensitivity and resolution, allowing for accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Pepstatin, by Merck Group (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Anti pan erk, by BD (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Ab2302, by Merck Group (1 mentions)
Immobilon membrane, by Merck Group (1 mentions)
Illustra tripleprep kit, by GE Healthcare (1 mentions)
Mes running buffer, by Thermo Fisher Scientific (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ab183127, by Abcam (1 mentions)
Ab37267, by Abcam (1 mentions)
Goat anti rabbit 800, by LI COR (1 mentions)
Ab92501, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
5 protocols using odyssey classic infrared imager
1
Immunoblot Analysis of Splenic NK Cells
2
Protein Expression Analysis in Vascular Fractions
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Proteins of interest were evaluated in vessels and parenchyma fractions. Samples were prepared with Laemmli sample buffer and boiled at 95°C for 4min. Molecular weight marker PageRuler Plus Prestained Protein Ladder ThermoFisher) was used to determine the protein of interest.
Proteins were then transferred to a nitrocellulose membrane (Bio-Rad) blocked with 5% milk/Tween-20-TBS (T-TBS) 0,1%. Membranes were incubated with primary antibodies, LOX-1
(1:1000 #ab60178 Abcam), Occludin (1:500 #71-500 Invitrogen), VCAM-1 (1:500 #sc-13160 were obtained by an Odyssey Classic infrared imager (LI-COR) and inverse band densities were then analyzed using ImageStudio 3.0 software (LI-COR). For parenchyma and vessel isolates, gels were imaged using the ChemiDoc XRS+ (BIO-RAD), and band intensity analyzed in ImageJ.
Proteins were then transferred to a nitrocellulose membrane (Bio-Rad) blocked with 5% milk/Tween-20-TBS (T-TBS) 0,1%. Membranes were incubated with primary antibodies, LOX-1
(1:1000 #ab60178 Abcam), Occludin (1:500 #71-500 Invitrogen), VCAM-1 (1:500 #sc-13160 were obtained by an Odyssey Classic infrared imager (LI-COR) and inverse band densities were then analyzed using ImageStudio 3.0 software (LI-COR). For parenchyma and vessel isolates, gels were imaged using the ChemiDoc XRS+ (BIO-RAD), and band intensity analyzed in ImageJ.
3
Neuronal Protein Immunoblotting Analysis
4
Protein Separation and Visualization
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Separation of proteins by non denaturing polyacrylamide gel was performed, as previously described [15 (link)]. Shortly after this, purified complexes were mixed as indicated, and resolved on nondenaturing polyacrylamide gradient gel (8−18%) at pH 8.8. Proteins were transferred on nitrocellulose membrane and visualized using a direct fluorescence Typhoon FLA 9500 biomolecular imager (GE Healthcare) at 555 nm or detected with an anti-myc mAb and a secondary antibody IRDye800-conjugated anti-mouse (Science-Tec) and visualized on an Odyssey Classic Infrared Imager (LI-COR).
5
Quantitative Western Blotting of FABPs
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Immunoblotting used primary antibodies recognising FABP1 (Abcam (AB76812; rabbit, 1:300), FABP2 (gift from Dr Satoshi Kaiura, Dainippon Sumi-tomo Pharma Co. Ltd., Osaka, Japan; mouse; 1:400), FABP3 (R&D Systems MAB1678; rabbit, 1:200), FABP4 (Abcam AB92501; rabbit, 1:1000), FABP5 (Abcam AB37267; rabbit, 1:200), GR (Abcam AB183127; rabbit, 1:400) or b-actin (Cell Signaling Technology 3700S; mouse, 1:5000). Primary antibodies were detected using the following fluorescent secondary antibodies: goat anti-mouse 680 (LI-COR 926-68070; 1:10,000), goat anti-rabbit 800 (LI-COR 926-32211; 1:10,000).
Proteins were resolved by SDS-polyacrylamide gel electrophoresis using precast 4-15% Mini-PROTEAN TGX gels (Bio-Rad) and transferred to 0.45-mm LF PVDF (low fluorescence polyvinylidene difluoride) membranes (Bio-Rad) using a Trans-Blot SD Semi-Dry Transfer Cell (for 75 min at 10 V; Bio-Rad). Membranes were blocked for 1 hour at room temperature (5% w/v BSA in PBS with 0.1% v/v Tween 20 [PBS-T]) and incubated with primary antibody overnight at 4°C (diluted in 1% w/v BSA). Membranes were washed, incubated with secondary antibody (diluted in PBS-T) for 1 hour at room temperature, and washed. Immunoreactivity was detected by fluorescence using the Odyssey Classic Infrared Imager (LI-COR Biosciences), with resolution set at 169 μm.
Proteins were resolved by SDS-polyacrylamide gel electrophoresis using precast 4-15% Mini-PROTEAN TGX gels (Bio-Rad) and transferred to 0.45-mm LF PVDF (low fluorescence polyvinylidene difluoride) membranes (Bio-Rad) using a Trans-Blot SD Semi-Dry Transfer Cell (for 75 min at 10 V; Bio-Rad). Membranes were blocked for 1 hour at room temperature (5% w/v BSA in PBS with 0.1% v/v Tween 20 [PBS-T]) and incubated with primary antibody overnight at 4°C (diluted in 1% w/v BSA). Membranes were washed, incubated with secondary antibody (diluted in PBS-T) for 1 hour at room temperature, and washed. Immunoreactivity was detected by fluorescence using the Odyssey Classic Infrared Imager (LI-COR Biosciences), with resolution set at 169 μm.
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