Tissue lysates were homogenized, lysed, and quantified as above, and then treated with an equal amount of SDS-PAGE loading buffer (62.5 mM Tris-Cl, pH 6.8, 20% glycerol, 7.5% SDS, 5% 2-mercaptoethanol, and 250 mM DTT) and denatured by heating 95°C. Proteins were then separated using an SDS-PAGE gel and transferred to a polyvinylidene Immobilon-FL membrane. Membranes were washed in PBS, then soaked in 5% milk + PBST to block nonspecific binding. The membranes were incubated in a primary rabbit α-human A3A/B/G antibody 1:1,000 (Brown et al.) and mouse α-tubulin 1:10,000 (Sigma Aldrich), or rabbit α-actin 1:5,000 (Sigma Aldrich) at 4°C overnight. Membranes were then washed in PBST six times for 5 minutes each, then incubated for one hour with secondary α-rabbit HRP (Cell Signaling Technology) and goat α-mouse 800 (LI-COR) at 1:10,000 dilutions supplemented with 0.02% SDS. These membranes were then washed 5 times in PBST and one time in PBS for 5 minutes each, then imaged using an Odyssey Classic scanner and Odyssey Fc imager (LI-COR)
Odyssey classic scanner
Manufactured by LI COR
The Odyssey Classic scanner is a high-performance fluorescence imaging system designed for a variety of applications. It is capable of detecting and quantifying near-infrared (NIR) fluorescent signals with exceptional sensitivity and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (3 mentions)
Mouse α tubulin, by Merck Group (2 mentions)
Rabbit α actin, by Merck Group (2 mentions)
α rabbit hrp, by Cell Signaling Technology (2 mentions)
Goat α mouse 800, by LI COR (2 mentions)
Odyssey fc imager, by LI COR (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Odyssey blocking buffer tbs, by LI COR (1 mentions)
Bca protein assay, by Promega (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by LI COR (1 mentions)
Fish gelatin, by Merck Group (1 mentions)
Image studio software, by LI COR (1 mentions)
Ripa buffer, by Rockland Immunochemicals (1 mentions)
Irdye 800cw donkey anti mouse igg, by LI COR (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Recombinant activin a, by R&D Systems (1 mentions)
9 protocols using odyssey classic scanner
1
Western Blot Analysis of Cellular Proteins
2
Extraction and Fractionation of Cellular Proteins
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Whole cell lysate was extracted in radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl, 1% NP-40, 0.5 % Na deoxycholate, 0.1 % Na dodecyl sulfate, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, and 1 X protease and phosphatase inhibitor cocktail (Halt, ThermoFisher Scientific #78440). Cytoplasmic and nuclear fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific #PI78835). The estimation of protein concentration was done using BCA protein assay (Promega #PI-23222, PI-23224). Samples were diluted using 1 X sample buffer (4 X stock, LI-COR #928–40004) with 100 mM dithiothreitol (DTT) (10 X stock, 1 mM, Sigma #D9779-5G). The protein was separated using 8–12% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with Odyssey TBS blocking buffer (LICOR-Biosciences #927–50003) for 45 min at room temperature, then incubated with primary antibodies (Key Resources Table) at least overnight at 4°C. IRDye 800CW and 680RD secondary antibodies (LI-COR Biosciences # 926–32211, # 926–68072) were diluted 1:10,000 in 0.1% TBS-Tween and imaged on the Odyssey Classic Scanner (LI-COR Biosciences).
3
Western Blot Protein Detection
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Sample aliquots of lysates containing ∼50–125 μg of protein were added to sample buffer supplemented with β-ME and denatured at 95°C for 10 min before electrophoresis on precast 4–12% gradient polyacrylamide gels (NuPAGE; Invitrogen, Carlsbad, CA) run at 125 mV for 2 h. Protein transferred to nitrocellulose membrane (iBlot; Invitrogen, Carlsbad, CA) was incubated at RT for 30 min with Odyssey blocking buffer (LI-COR, Lincoln, NE) and subsequently incubated with targeted primary antibody. Excess antibody was removed in four consecutive washed with Tween-20 buffer (Tris-HCl 30 mM, pH 7.4, NaCl 150 mM, and Tween-20 0.1%). Bound antibody was detected with corresponding secondary antibody conjugated to either IRDye680 or IRDye800 as indicated and analyzed in an Odyssey Classic scanner (LI-COR, Lincoln, NE).
4
Western Blot Analysis of Epithelial Proteins
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Cultured mCCDcl1 cells were directly lysed with radioimmunoprecipitation assay buffer, whereas organ samples were lysed with radioimmunoprecipitation assay and mechanical homogenization using a tissue lyser. Proteins were quantified by BCA assay, and equal amounts of protein were separated by SDS–PAGE, then transferred to nitrocellulose membranes. After protein visualization by Ponceau S dye and blocking with TBS/Tween buffer containing 5% milk, membranes were incubated at 4°C overnight with primary antibodies (Table 3 ), then 1 h with DyLight anti-mouse and anti-rabbit secondary antibodies (#35519 and #SA5-10036, 1:5,000; Invitrogen). Blots were visualized using the Odyssey classic scanner (LI-COR Biosciences), quantified using Image Studio Lite software, and normalized to tubulin expression. For Prss8, St14, ZO-1, occludin, and E-cadherin proteins, secondary anti-rabbit, anti-mouse, and anti-sheep antibodies (1:10,000; Amersham Biosciences) were detected by chemiluminescence (SuperSignal West Pico; Thermo Fisher Scientific).
5
Electroblotting and Ubiquitination Analysis
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Nitrocellulose membranes (BIO-RAD 1620213 or 1704159) were used for the electroblotting of proteasomal substrates. PVDF (BIO-RAD 1704156) was used for the blotting of free Ub and was autoclaved in water after blotting [77 (link)]. After blocking in Odyssey Blocking Buffer (PBS, LI-COR 927), membranes were incubated sequentially with primary and secondary antibodies in Odyssey Blocking Buffer mixed with equal volume of PBS and Tween 20 at 0.1% (v/v). After each incubation, membranes were washed in PBS plus 0.1% (v/v) of Tween 20. Tween 20 was removed by rinsing in PBS before detecting the fluorescence of secondary antibodies using a LI-COR Odyssey Classic Scanner. The fluorescence of protein bands was quantified by Odyssey Application Software while the ubiquitination profiles were quantified in ImageQuant TL by 1D Gel Analysis.
6
Protein Extraction and Western Blot Analysis
7
Western Blot Analysis of Endothelial Proteins
8
Western Blot Analysis of APOBEC Proteins
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Tissue lysates were homogenized, lysed, and quantified as above, and then treated with an equal amount of SDS-PAGE loading buffer (62.5 mM Tris-Cl, pH 6.8, 20% glycerol, 7.5% SDS, 5% 2-mercaptoethanol, and 250 mM DTT) and denatured by heating at 95°C. Proteins were then separated using an SDS-PAGE gel and transferred to a polyvinylidene Immobilon-FL membrane. Membranes were washed in PBS, then soaked in 5% milk + PBST to block nonspecific binding. The membranes were incubated in a primary rabbit α-human A3A/B/G antibody 1:1,000 (Brown et al.) and mouse α-tubulin 1:10,000 (Sigma Aldrich), or rabbit α-actin 1:5,000 (Sigma Aldrich) at 4°C overnight. Membranes were then washed in PBST six times for 5 min each, then incubated for 1 h with secondary α-rabbit HRP (Cell Signaling Technology) and goat α-mouse 800 (LI-COR) at 1:10,000 dilutions supplemented with 0.02% SDS. These membranes were then washed 5 times in PBST and one time in PBS for 5 min each, then imaged using an Odyssey Classic scanner and Odyssey Fc imager (LI-COR).
9
Validating Activin B Antibody Specificity
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To confirm that the primary antibody detected activin b C in mouse ovary protein extract and did not cross-react with activin b A , western blotting was performed according to the methods described by Marino et al. (2015) (link). Briefly, frozen WT mouse ovaries were homogenised in RIPA buffer containing protease inhibitors, centrifuged at 13 000g for 15 min at 48C and 40 mg protein subjected to electrophoretic separation on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) under nonreducing conditions. Recombinant activin A, activin AC and activin C (R&D Systems; 10 ng each) were separated on a 15% gel under reducing conditions. Following transfer to nitrocellulose membranes and blocking with Odyssey Blocking buffer (LI-COR Biosciences), membranes were incubated overnight with a 1 : 1000 dilution of anti-activin b C antibody. Secondary antibody detection was performed using IRDye 800CW goat antimouse IgG1 (LI-COR Biosciences) and imaged with an Odyssey Classic scanner and Image Studio Lite software (LI-COR).
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