Whole liver was immediately frozen using clamps pre-cooled to the temperature of liquid nitrogen and stored at −80° for Western blot analysis. Briefly, samples were analyzed for protein content, and separated on 4%–12% Bis-Tris gels at 150 volts for 1.5 h. Transfer was for 2 h at 30 volts on ice. Blots were incubated with primary antibody to GR (1:1000; Cell Signaling Technology, Danvers, MA, USA), PEPCK (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), 11β-HSD1 (1:1000; Abcam Cambridge, MA, USA), G6Pase α (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and G6Pase β (1:200; Santa Cruz Biotechnology Inc.) overnight at 4°C. To probe for actin, blots were incubated with anti-actin primary antibody (1:5000; EMD Millipore, Billerica, MA, USA) for 1 h at room temperature. After washing, blots were incubated with secondary antibody, anti-rabbit Immunoglobulin G (IgG) (heavy and light chains) DyLight (1:100,000; Cell Signaling Technology) and simultaneously with anti-mouse IgG (H + L) DyLight (1:15,000; Cell Signaling Technology), for 1 h at room temperature. Images of membranes were obtained with the abundance of all proteins of interest normalized to actin, which was the internal control. Band density was analyzed using Odyssey-Clx (LI-COR, Lincoln, NE, USA) and Image Studio (LI-COR).
Anti actin primary antibody
Manufactured by Merck Group
Sourced in United States
The Anti-actin primary antibody is a laboratory reagent used in various scientific and research applications. It is designed to specifically bind to and detect the actin protein, which is a key structural component of eukaryotic cells. The antibody can be utilized in techniques such as Western blotting, immunohistochemistry, and immunofluorescence to visualize and analyze the presence and distribution of actin within biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg h l dylight, by Cell Signaling Technology (2 mentions)
Dylight, by Cell Signaling Technology (2 mentions)
Image studio, by LI COR (2 mentions)
Odyssey clx, by LI COR (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pepck, by Santa Cruz Biotechnology (1 mentions)
11β hsd1, by Abcam (1 mentions)
G6pase α, by Santa Cruz Biotechnology (1 mentions)
Tissue pe lb, by G Biosciences (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Pparα, by Abcam (1 mentions)
Anti gata 3, by Santa Cruz Biotechnology (1 mentions)
Hdac family antibody set, by Abcam (1 mentions)
Anti t bet, by Santa Cruz Biotechnology (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Saf83, by Cayman Chemical (1 mentions)
Pierce cell surface protein biotinylation and isolation kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti actin primary antibody
1
Quantitative Analysis of Hepatic Protein Expression
2
Liver and Kidney Protein Analysis using Western Blotting
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Liquid homogenization of the liver and kidney samples was performed, and samples were stored at −80 °C. Briefly, samples were analyzed for protein content (Tissue PE LB, G-Biosciences, St-Louis, MO, USA) and separated on 4–12% Bis-Tris gels at 150 volts for 1.5 h. Dry transfer was performed using the iBlot2 Dry Blotting system (Thermo Fisher Scientific, Waltham, MA, USA). Blots were incubated with primary antibody for OCTN2 (1:1000; Abcam Cambridge, MA, USA), BBH (1:1000; Abcam, Cambridge, MA, USA), PPAR-α (1:1000; Abcam Cambridge, MA, USA), and GAT2 (1:1000; Abcam, Cambridge, MA, USA) overnight at 4 °C. To probe for actin, blots were incubated with anti-actin primary antibody (1:5000; EMD Millipore, Billerica, MA, USA) for 1 h at room temperature. After washing, blots were incubated with the secondary antibody anti-rabbit immunoglobulin G (IgG) (heavy and light chains) DyLight (1:100,000; Cell Signaling Technology, Danvers, MA, USA) and, simultaneously, with anti-mouse IgG (H + L) DyLight (1:100,000; Cell Signaling Technology) for 1 h at room temperature. Images of membranes were obtained, with the abundance of all proteins of interest normalized to actin, which was the internal control. Band density was analyzed using Odyssey-Clx and Image Studio (LI-COR, Lincoln, NE, USA).
3
Western Blot Analysis of Transcription Factors
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The right upper lung tissues were homogenized in the presence of protease inhibitors, and the protein concentrations were determined using NE-PER® nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, USA). The total proteins (50 μg) were loaded onto SDS-PAGE gels. After electrophoresis at 120 V for 90 min, the separated proteins were transferred to polyvinylidene difluoride membranes using a wet transfer method (250 mA for 90 min) [21 (link)]. Nonspecific sites were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h, and the membranes were then incubated overnight at 4°C with anti-GATA-3, anti-T-bet (Santa Cruz Biotechnology Inc., USA), or anti-HDAC1–11 (HDAC Family Antibody Set; BioVision, USA) primary antibodies. Horseradish peroxidase-conjugated anti-rabbit IgG was used to detect binding of the primary antibodies. The membranes were stripped and reprobed with an anti-actin primary antibody (Sigma-Aldrich, USA) to verify equal protein loading in each lane. The binding of the specific antibodies was visualized by exposure to photographic film. The densities of the stained bands for T-bet, GATA-3, and HDAC1–11 relative to the staining band for β-actin were quantified with Quantity ONE densitometry software (PDI Imageware Systems, USA).
4
Western Blot Analysis of Kinesin-1 Proteins
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Fresh kidney tissues were washed with cold PBS and lysed in RIPA buffer (50 mM Tris-Cl (pH7.4), 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 1mM EDTA, 0.1% SDS, 0.01% Sodium Azide). The protein concentrations of lysates were determined using a BCA Protein Assay Kit (Thermo). Equal amounts of protein samples were loaded onto SDS-PAGE gels for electrophoresis and subsequent Western blot analysis. The Kif5b-specific antibody used has been described elsewhere [24 (link)]. The blots were incubated with anti-Kif5b primary antibody (1:2000, against synthesized peptide FDKEKANLEAFTADKDIA), anti-Kif5a (1:100, against synthesized peptide NGNATDINDNRSDLPC), anti-Kif5c (1:100, against synthesized peptide SAKDQKSLEPC) (S1 Fig ) and anti-actin primary antibody (1:3000, Sigma) at 4°C overnight, followed by incubation with HRP-(horseradish peroxidase)-conjugated secondary antibodies at room temperature for 1 hour. An enhanced chemiluminescence kit (Pierce) was used for detection of the immunoreactive bands.
5
Western Blot Analysis of Prion Protein
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Samples were run on 12% or 14% Tris-glycine gels using a Bio-Rad system and transferred to polyvinyl difluoride (PVDF; Millipore) membranes. Blots were immediately incubated with PrP antibody Sha31 (Spi-Bio Inc; 1/30,000 in 0.5% TBST) or were blocked with 5% skim milk in 0.1% TBST for 1 h at room temperature and incubated with primary antibody at 4°C overnight (PrP antibodies: 9A2 1/4,000, 12B2 1/8,500, SAF83 (Cayman), VRQ61 1/5,000, and 2D6 1/10,000, 1A6 1/5,000). 9A2 and 12B2 were gifts from Dr. Jan Langeveld (Langeveld et al, 2006 (link)), VRQ61 and 2D6 from Dr. Human Rezaei (Moudjou et al, 2004 (link)), and 1A6 was produced in-house. Membranes were subsequently incubated with secondary antibody at 1/10,000 (Bio-Rad) for 2 h at room temperature and visualized using ECL (Pierce). After stripping, membranes were incubated in anti-actin primary antibody (Sigma; 1/5,000).
6
Isolation and Analysis of Membrane Proteins
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For isolation of membrane proteins, the Pierce Cell Surface Protein Biotinylation and Isolation Kit (ThermoFisher) was used according to the manufacturer's protocol. In brief, cells were cultured for 48 h and transfected with either the WT or one of the mutant plasmids. Cells were biotinylated, lysed and isolated by binding to probed agarose beads 24 h after transfection. After elution, the proteins were prepared for Western blot analysis. Western blots were performed and analysed as described above. A monoclonal mouse anti-actin primary antibody (1:30,000, A5441, Sigma-Aldrich) was used as control.
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