Tissues were lysed in 1× lysis buffer (Cell Signaling Technology, #9803) with protease inhibitor cocktail (EMD Biosciences, 539131). Mouse monoclonal IDH2 antibody (Abcam, #ab55271) was used at 1 μg/mL dilution, rabbit monoclonal β-actin antibody (Cell Signaling Technology, #4970) was used at 1:2000 dilution, GYS1 antibody (Cell Signaling Technology, #3893) was used at 1:1000 dilution, and UGP2 antibody (Abcam, ab157473) was used at 1:1000 dilution.
β actin rabbit monoclonal antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The β-actin rabbit monoclonal antibody is a laboratory reagent used to detect and quantify the expression of the β-actin protein, a ubiquitously expressed cytoskeletal protein. It can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to analyze the presence and levels of β-actin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rabbit monoclonal gfp antibody, by Abcam (1 mentions)
Goat anti rabbit igg conjugated to hrp, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Ecl western blotting analysis system, by GE Healthcare (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Hrp linked goat anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence detection system, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Tnf α, by Cell Signaling Technology (1 mentions)
Supersignal west pico enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Abcam (1 mentions)
15 protocols using β actin rabbit monoclonal antibody
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Viral Proteins
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Cells were lysed in RIPA lysis buffer [150 mM NaCl, 1% Non-ionic detergent, 1% Sodium deoxycholate, 0.1%SDS, (Biomax, Republic of Korea)]. About 20 μg of proteins per sample were separated by SDS-PAGE using a 10%polyacrylamide gels and transferred to nitrocellulose membranes (Cytiva, USA). Membranes were blocked with 10% (w/v) non-fat dry milk in TBST buffer [100 mM Tris (pH 7.6), 150 mM NaCl, 0.1% (w/v) Tween-20] for 1 h at room temperature. Membranes were incubated with a 1:1,000 dilution of rabbit polyclonal anti-Gag antibody, 1:500 dilution of rabbit polyclonal anti-Env antibody, 1:5,000 dilution of rabbit monoclonal GFP antibody (Abcam, UK), and 1:5,000 dilution of rabbit monoclonal β-actin antibody (Cell Signaling Technology, USA) at 4°C overnight. The membranes were then incubated with a 1:10,000 dilution of secondary antibody, goat anti-rabbit IgG conjugated to HRP (BioRad, USA), for 1 h at room temperature. The chemiluminescent signal was visualized by exposing the blots to a chemiluminescence imaging system, E-blot (e-BLOT Life Science, China). Rabbit polyclonal anti-Env antibody was designed to be specific for PFV Env-SU domain and was custom-produced by AbClon (Korea).
3
Antibody Preparation and Characterization
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Polyclonal rabbit anti-Flag (F7425), polyclonal rabbit anti-HA (H6908), and monoclonal mouse anti-HA (H3663) were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal rabbit β-actin antibody was purchased from Cell Signalling (4967 S) (Danvers, MA).
4
Quantitative Western Blot Analysis
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Protein was extracted through radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and protease inhibitors (Roche Diagnostics). Total protein was measured using a bicinchoninic acid (BCA) assay. The protein (30 µg per lane) was separated by 10% polyacrylamide gel electrophoresis and electro-blotted to polyvinylidene fluoride membranes. After blocking with 5% nonfat milk in TBST for 2 h at room temperature, the membranes were subsequently incubated overnight at 4˚C with the indicated antibodies (1:1,000 dilution), followed by incubation with secondary antibodies (1:2,000 dilution) for 2 h at room temperature. The primary antibodies included rabbit polyclonal to spred2 antibody (cat. no. ab153700; Abcam), monoclonal rabbit p62 antibody (cat. no. 8025; Cell Signaling Technology, Inc.), monoclonal rabbit LC3-I antibody (cat. no. 4599; Cell Signaling Technology, Inc.), monoclonal rabbit LC3-II antibody (cat. no. 3868; Cell Signaling Technology, Inc.), monoclonal rabbit β-actin antibody (cat. no. 4970; Cell Signaling Technology, Inc.). The secondary antibodies were HRP-conjugated goat anti-rabbit IgG antibodies (cat. no. 7074; Cell Signaling Technology, Inc.). The gray values were measured using ImageJ software (National Institutes of Health).
5
Protein Expression Analysis of PBMC
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Cultures of PBMC were harvested and washed in ice-cold PBS three times. Proteins were isolated from the cells with RIPA buffer (Thermo Scientific cat # 89901). Total protein was quantitated using Pierce® BCA protein assay kit- reducing agent compatible (Thermo scientific). Equal amounts of protein (5μg) were heated (70°C for 10 minutes) in the NuPAGE® LDS sample buffer and NuPAGE®reducing agent and separated on NuPAGE® Bis-Tris Gel (4–12%) (Life technologies) and blotted onto Amersham Hybond ECL Nitrocellulose membrane. Cellular proteins were detected with the primary mouse monoclonal antibody raised against recombinant 3PGDH of human origin (Santa Cruz Biotechnology, Inc., Cat # sc-100317), rabbit polyclonal antibody raised against synthetic PSAT1 peptide of human origin (Santa Cruz Biotechnology, Inc., Cat # sc-133929) and β-Actin rabbit monoclonal antibody (Cell Signaling). Proteins were visualized using an ECL western blotting analysis system (GE Healthcare).
6
IFN-β-Induced STAT1 Phosphorylation Assay
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HeLa cells were stimulated with 1000 IU of IFN-β and cells were collected at determined time points. Cells were lysed in lysis buffer (1% 50 mM Tris-HCl, 150 mM NaCl, Triton-X 100) with protease inhibitors for 30 min at 4 °C. Protein estimation was performed by the BCA method and equal amounts (20 µg) of protein were separated on a 12% gel by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred from the gel to polyvinylidene difluoride (PVDF) membrane and probed with phospho-STAT1 (Y701) or β-actin rabbit monoclonal antibody (Cell signalling, Beverly, MA, USA) with gentle shaking at 4 °C overnight. Respective HRP-conjugated anti-rabbit antibodies were used as secondary antibodies (Cell signalling, Beverly, MA, USA). Detection was performed using ECL Plus Western blot detection reagents (Pierce, ThermoFisher) following the manufacturer’s instructions. Images, as presented in Supplementary Figure S1 , were acquired on a Amersham imager 680 (GE, United Kingdom) machine.
7
Investigating MAPK Signaling Pathways
8
Evaluating Renal Inflammatory Markers
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The expression of TNF-α and COX-2 in renal tissues was evaluated using western blot analysis. Protein extracts were obtained by homogenizing samples in ice-cold lysis buffer, followed by two cycles of centrifugation at 15700xg for 15 min at 4°C. Protein concentrations were determined by the Bradford assessment. An equal amount of protein from each sample was electrophoresed on 12% SDS polyacrylamide gels and then blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 2% skim milk prepared in TBST (Tris-buffered saline and 0.1% Tween 20) for 75 min at room temperature and then incubated overnight at 4°C with COX-2 (ABR-Affinity BioReagents, Golden, CO, USA) and TNF-α (Cell Signaling, Danvers, MA, USA) antibodies. Membranes were then washed three times with TBST, incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Danvers, MA, USA) for 60 min at room temperature and finally visualized using an enhanced chemiluminescence detection system (GE Healthcare, Amersham, UK). Equal loading was confirmed by stripping the immunoblots and reprobing with β-actin rabbit monoclonal antibody (Cell Signaling). At the end, the COX-2 and TNF-α protein bands were assessed semiquantitatively by densitometric evaluation of scanned X-ray films using ImageJ software, and normalized against β-actin bands.
9
Western Blot Analysis of Nectin-1 in Mouse Brain Lysates
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Mouse brains were minced and lysed in Pierce RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Tissue lysate was collected by centrifugation and quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). An equal amount of lysate was boiled in loading buffer and subjected to SDS-PAGE. Subsequently, the separated protein in gel was transferred onto the PVDF membrane, which was then blocked in 5% milk and incubated with anti-nectin1 mouse monoclonal antibody (sc-21722, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight. The membrane was washed and further incubated with HRP-conjugated goat-mouse IgG secondary antibody. After thorough washing, the target protein was imaged using a SuperSignal West Pico enhanced chemiluminescence kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The same blots were probed with a β-actin rabbit monoclonal antibody (13E5, Cell Signaling Technology, Danvers, MA, USA) as a loading control.
10
Western Blot Analysis of LonP1 Protein
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Proteins were separated by SDS-PAGE under reducing conditions and then semi-dry transferred onto polyvinylidene difluoride (PVDF) membranes for 7 min using a Bio-Rad Turbo Transfer system. Then, PVDF membranes were blocked with 5% milk in TBST. Western-blot analysis against LonP1 was carried out using a rabbit polyclonal anti-LonP1 antibody (Abcam, Cambridge, MA, USA, #ab224316, diluted 1:1,000, overnight at 4 °C) with β-actin rabbit monoclonal antibody (Cell Signaling Technology, #8457, employed 1:1,000) as a loading control. Following incubation with HRP-conjugated goat antirabbit secondary antibody (Abcam, Cambridge, MA, USA, #ab6721 at 1:6000 dilution for 75 min), HRP-conjugated proteins were detected with the SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA, #34577) and visualized using the Bio-Rad (Hercules, CA, USA) ChemiDoc imaging system.
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