Aliquots from fat pad homogenates were used for protein (Bicinchoninic Acid Kit, Thermo Scientific, Waltham, MA) measurements, and Western blots. For Western blot analyses, tissue lysates were separated on 10% acrylamide gel under reducing conditions. After transfer to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Billerica, MA), the membranes were stained with primary antibodies against UCP1 (Abcam, Cambridge, MA), PGC-1α (Abcam, Cambridge, MA), and β-actin (Cell Signaling, Danvers, MA) and then detected with the appropriate secondary antibody (LI-COR, Lincoln, NE). Proteins were analyzed using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). Images were quantified by ImageJ software.
Appropriate secondary antibody
Manufactured by LI COR
Sourced in United States
Appropriate secondary antibodies are laboratory reagents used to detect and visualize primary antibodies bound to target proteins. They function by specifically binding to the constant regions of primary antibodies, enabling the detection and amplification of the primary antibody signal. These secondary antibodies are conjugated with various reporter molecules, such as fluorescent dyes or enzymes, to facilitate visualization and quantification of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (3 mentions)
Raf rbd beads, by Cytoskeleton (2 mentions)
Primary antibody to nras, by Santa Cruz Biotechnology (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Blocking buffer, by LI COR (2 mentions)
Odyssey system, by LI COR (2 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Pgc 1α, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor tablet, by Roche (1 mentions)
Mem per plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Odyssey clx system, by LI COR (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Mini beadbeater 8, by Biospec (1 mentions)
Integrin β3, by Cell Signaling Technology (1 mentions)
13 protocols using appropriate secondary antibody
1
Western Blot Analysis of Adipose Tissue
2
Protein Extraction and Western Blot Analysis
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For total protein, heart tissues and NRVMs were lyzed in radio immunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail tablet and phosphatase inhibitor tablet (Roche, Germany). Protein content was measured according to the Pierce BCA protein assay kit (Sangon Biotech, China). For membranous protein, membrane protein in heart tissues and NRVMs were separated by the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher, United States) following the manufacturer’s protocol (Phuchareon et al., 2015 (link)). Then equal protein lysates were resolved electrophoretically on denaturing SDS-polyacrylamide gels, and transferred to 0.22 μm nitrocellulose membranes. After blocking in 5% milk in TBST, membranes were probed with the following primary antibodies: DRD4 (Sigma, Germany), cleaved caspase 3 (CST, United States), PMCA (CST, United States), GLUT4 (CST, United States), p-PI3K (CST, United States), PI3K (CST, United States), p-AKT (CST, United States), AKT (CST, United States), ATP1A1 (CST, United States) and GAPDH (Abcam, United Kingdom). The appropriate secondary antibody (LI-COR, United States) was used. The ATPase alpha-1 (ATP1A1) and plasma membrane-type Ca2+-ATPases (PMCA) as the loading control of membrane protein, while GAPDH as loading control of the total protein. The results were tested by Odyssey system and quantified by ImageJ.
3
Determination of NRas Activation
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NRas activation was determined as reported previously [12 (link)]. Briefly, lysates were cleared, and diluted to equal protein concentration as confirmed by Bradford Analysis. Raf-RBD beads (Cytoskeleton) were added to each sample and incubated at 4°C, collected by centrifugation, washed, and resuspended in Laemmli Sample buffer (Bio-Rad). The samples were then run on a Tris-HCl gel, transferred to a nitrocellulose membrane, and immunoblotted with a primary antibody to NRas (Santa Cruz) and appropriate secondary antibody (LiCor). The membrane was visualized using LiCor’s Odyssey infrared imaging system. Ponceau Red staining for total GST-RBD was used as loading control.
4
Immunoprecipitation and Immunoblotting for EFhd2
5
NRAS Activation Analysis Protocol
6
Protein Expression Analysis in Yeast Mutants
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For expression analysis, overnight cultures of Wt and mutants in EMM media lacking uracil were transferred to 50 ml and further grown to A600 of 0.8–1.0, washed with water, residual liquid aspirated and 200 μl of lysis buffer (50 mM HEPES (pH 7.3), 250 mM KCl, 5 mM EDTA, 2.5 mM DTT) was added. Glass beads (0.5 mm) were added and cells disrupted in mini-beadbeater-8 (Biospec) at 4 x 45 seconds keeping in ice for 1 min between intervals. Samples were then spun in tabletop centrifuge for 20 min at top speed, supernatant transferred to a new tube and protein concentration determined. 25 μg total protein was resolved in 4–12% Nu-PAGE gel (Life Technologies), transferred to nitrocellulose membrane. The blot was blocked for one hour with 5% non-fat milk in PBS. Monoclonal anti-flag (Sigma, #F1804) was used at 1:2000 in blocking buffer with 0.05% NP-40. Anti-Tubulin (Sigma, #T5168) was used at 1:5000. After one hour the blot was washed four times with PBS-NP-40. Appropriate secondary antibodies (LI-COR) of different fluorescence were used at 1:10,000 in 5% milk solution in PBS-NP-40 for one hour. The blot was washed with PBS-NP-40 four times and scanned using LI-COR Odyssey Clx system and the images processed with ImageStudioLite Software.
7
SDS-PAGE and Western Blot Analysis
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BMDM lysates were separated by 8 and 15% SDS-PAGE, and then transferred onto PVDF membranes. After blocking for 1 h at room temperature with blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), blots were incubated with primary antibody at 4 °C overnight, followed by incubation with appropriate secondary antibodies (LI-COR Biosciences) for 1 h. Protein bands were detected using the Odyssey System from LI-COR Biosciences and the intensity of each band was quantified using ImageJ version 1.50i. The intensity of target protein band was normalized with reference protein band and calculated for the fold changing.
8
Integrin and Connexin Protein Analysis
9
Quantitative Western Blot Analysis
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The supernatant of PLF or cell culture medium was concentrated 20 times by Ultracel-3 membrane 3 kDa, and the BMDM lysates were separated by 8% and 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto the PVDF membranes. After blocking for 1 h at room temperature with blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), the blots were incubated with the primary antibody at 4 °C overnight, followed by incubation with appropriate secondary antibodies (LI-COR Biosciences) for 1 h. Protein bands were detected using the Odyssey System from LI-COR Biosciences, and the intensity of each band was quantified using ImageJ version 1.50i. The intensity of the target protein band was normalized with a reference protein band and calculated for the fold changing.
10
Western Blot Analysis of Ovarian Proteins
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Total protein was extracted from ovaries with lysis buffer (Beyotime, Haimen, China) and Bullet Blender (NY, USA). Protein concentration was determined using bicinchoninic acid (BCA) protein assay reagent. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, BM1623; Boster, Beijing, China) was used as the loading control. Samples were separated by 12% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride membranes (Millipore, USA). After blocking in 5% non-fat milk, membranes were immunoblotted with the following antibodies: ECR, Br (both from Developmental Studies Hybridoma Bank, USA). Kr-h1 (Santa Cruz Biotechnology, Santa Cruz, USA). Next, the membranes were incubated with appropriate secondary antibodies (Li-Cor, Lincoln, NE, USA) for 1 h at room temperature (31 (link)). Finally, chemiluminescence-based visualization and analysis of protein bands were conducted using a FluorChem imaging system (ProteinSimple, San Jose, CA, USA).
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