Protein levels from cell lysates or mouse lung homogenates were measured by western blotting using anti-ELF1 antibody (1:5000, Santa Cruz for murine Elf1; 1:5000, Bethyl labs for human ELF1), anti-IRF1 antibody (1:1000, Cell Signaling), anti-STAT1-antibody (1:1000, Cell Signaling), anti-STAT3 antibody (1:2000, Cell Signaling), anti-actin antibody (1:1000, Thermo Fisher). Actin was used as housekeeping control using anti-actin-HRP antibody (1:1000, Thermo Fisher) or anti-actin antibody (1:1000, Thermo Fisher). Relative band intensities were determined with ImageJ.
Anti actin antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The anti-actin antibody is a laboratory reagent used to detect and quantify the presence of the actin protein in biological samples. Actin is a ubiquitous cytoskeletal protein found in all eukaryotic cells and plays a crucial role in various cellular processes. The anti-actin antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to identify and analyze actin expression and distribution within cells and tissues.
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Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Anti his tag antibody, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Bm chemiluminescence blotting substrate pod, by Roche (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by LI COR (1 mentions)
Anti erk1 2 137f5, by Cell Signaling Technology (1 mentions)
Anti phospho p38 d3f9, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2 d13.14.4e, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg conjugated to irdye800cw, by LI COR (1 mentions)
Butylated hydroxyanisole bha, by Merck Group (1 mentions)
Mini protean tgx precast gradient gel, by Bio-Rad (1 mentions)
Las 3000 analyzer, by GE Healthcare (1 mentions)
Anti nrf2 antibody, by Active Motif (1 mentions)
Horseradish peroxidase conjugated species specific secondary antibodies, by GE Healthcare (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Anti α tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Prism 8, by GraphPad (1 mentions)
10 protocols using anti actin antibody
1
Western Blot Analysis of Key Transcription Factors
2
Quantification of Recombinant Protein Binding
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Cells were treated with rHP1286 (1 μg/ml) for 15 min. Unbound protein was removed by washing with PBS. Cells were further incubated with an anti-His-tag antibody (Thermo fisher) (1:1,000 dilution) in FACS buffer (2% BSA in PBS) for 1 h on ice. After incubation, cells were washed twice with FACS buffer and stained with an Alexa 488-conjugated anti-mouse IgG antibody (Molecular Probes) (1:5,000 dilution) for 30 min on ice, followed by washing with FACS buffer. Binding of HP1286 was analyzed by flow cytometry using an LSRFortessa flow cytometer. FlowJo software (Tree Star, USA) was used for the data analysis. Cell lysates from untreated or rHP1286-treated samples were immunoblotted with anti-His tag antibody. Anti-actin antibody (Thermo Scientific) (1:5,000 dilution) was used to confirm equal loading.
3
Western Blot Analysis of MAF Protein
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MAF protein levels in both miR-486-3p/NegCTR mimic-transfected and in LMAFvar2IDN/LXIDN-transduced CD34+ cells were detected by western blot analysis. Briefly: cells were harvested, washed twice with ice-cold PBS and lysed (5 × 105 cells/20 μl of lysis buffer) in 50 mM Tris (tris(hydroxymethyl)aminomethane)-Cl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 10 mM KCl, 1 mM EDTA, 20 mM NaF, 0.25% Na doexycholate, 5 mM dithiothreitol (DTT) and protease inhibitors (Complete, catalog #1697498, Roche). Total cellular lysates (15 μg for each sample) were loaded onto 12.5% SDS-polyacrylamide gel and blotted on nitrocellulose membrane. To control loading and transfer, after transfer the membranes were stained by Red Ponceau. Membranes were then preblocked in blocking solution, 5% milk in 0.1% TBST for 1 h at room temperature (RT), incubated with primary antibodies (1 : 2000 dilution of rabbit monoclonal anti-MAF antibody (1 : 2000 dilution, catalog #181188, Abcam, Cambridge, UK; www.abcam.com ), at 4 °C overnight of rabbit polyclonal anti–actin antibody (1 : 5000 dilution, Thermo Fisher Scientific Inc., Waltham, MA, USA, catalog #PA1-16889) for 1 h at RT. After three washes with TBST, blots were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1 : 1000 dilution, Thermo Fisher Scientific Inc.) for 1 h at RT and revealed by BM Chemiluminescence Blotting Substrate (POD) (Roche).
4
Cell Lysate Preparation and Immunoblotting
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The method used for cell lysate preparation and immunoblotting were described previously (Pathak et al., 2013 (link)). Briefly, cell lysates were prepared by the addition of SDS-PAGE sample reducing buffer to cells after washing twice with PBS, followed by heating at 95°C for 10 min. Immunoblotting was performed using anti-phospho ERK1/2 (D13.14.4E) (1:1,000 dilution), anti-phospho-p38 (D3F9), anti-phospho-JNK (81E11), and anti-ERK1/2 (137F5) (1:1,000 dilution) antibodies from Cell Signaling Technology. Anti-phospho-c-Fos (Thr325) antibody was from Bioss antibodies. An anti-actin antibody (Thermo Scientific) (1:5,000 dilution) was used to confirm equal loading. The secondary antibodies included goat anti-rabbit IgG and goat anti-mouse IgG conjugated to IRdye800CW (Li-COR) (1:10,000 dilution). Band intensities were quantified using the ImageJ software, and actin was used to normalize the total amount of protein loaded in each well.
5
NRF2 Activation in Mouse Heart Tissue
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Heart tissues were homogenized and lysed with RIPA buffer containing 10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate with protease and phosphatase inhibitor cocktails. Lysates were centrifuged at 20,000 g for 40 min at 4 °C and the supernatants were recovered. Total protein concentrations in the supernatants were measured using a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Scientific). For immunoblot analysis, extracted protein samples were separated on 7.5% Mini-PROTEAN TGX precast gradient gels (Bio-Rad) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% FBS in Tris-buffered saline plus 0.05% Tween and incubated overnight at 4 °C with an anti-NRF2 antibody (1:1000; Active Motif) and an anti-actin antibody (1:5000; Thermo Fisher Scientific) as a loading control. Primary antibodies were detected with horseradish peroxidase-conjugated species-specific secondary antibodies (GE Healthcare) and ECL plus (Thermo Fisher Scientific) using a LAS 3000 analyzer (GE Healthcare). Immunoblot band intensities were measured using NIH ImageJ software63 (link). Butylated hydroxyanisole (BHA; Sigma-Aldrich) was administered intraperitoneally to male mice at 8 weeks of age at a dose of 350 mg/kg in corn oil. Uncropped scans of blots are shown in Supplementary Fig. 14a, b, d .
6
Quantifying Cytoskeletal Proteins in Yeast
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Whole yeast protein extracts were obtained as described [82 (link)]. Actin and tubulin were visualized on an 8% SDS-PAGE with the anti-actin antibody (Thermo Scientific, Waltham, MA, USA) or anti-α-tubulin antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The densitometry of the bands was determined using Image Lab 6.1 (BioRad, Hercules, CA, USA) and quantification using GraphPad Prism8 (Graph-Pad Software, Inc., Boston, MA, USA).
7
Quantitative Proteomic Analysis by Western Blot
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For western blotting analysis, protein samples from TMT-based quantitative proteomics analysis were separated on SDS-PAGE and transferred onto PVDF membrane. After blocking with 5% milk in TBST (0.1% Tween-20) for 1.5 h, the membrane was incubated with anti-LCY (Agrisera Cat# AS132709), anti-PsaD (Agrisera Cat# AS09461), and anti-PsbO (Agrisera Cat# AS06142-33) polyclonal antibodies (1:5000 dilution) for 1 h at room temperature. Protein levels were visualized using ExPlus ECL chemiluminescence detection kit (Zomanbio Cat# ZD310) and detected by Tanon-5200 system (Tanon). Anti-Actin antibody (Thermo Fisher Cat# PA511571) was used as internal control.
8
Western Blot Analysis of Cellular Proteins
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Oocytes were lysed in RIPA buffer containing 1× protease inhibitor. Protein samples were boiled and separated by SDS-PAGE and then electrically transferred onto PVDF membrane. After transfer, the membranes were blocked in 1× TBST containing 5% skimmed milk or 3% BSA for 2 h at room temperature, followed by incubation with indicated primary antibodies overnight at 4 °C. The following antibodies were used: anti-actin antibody (Thermo Fisher Scientific), anti-EF2 antibody (Cell Signaling Technology, MA, USA), anti-HS90A antibody (Abcam, MA, USA), anti-RACK1 antibody (Abcam) and anti-COX IV antibody (Cell Signaling Technology). After washing in 1× TBST for three times, the membranes were incubated with 1:1000 dilution of HRP-conjugated secondary antibody for 1 h. For biotin-labeled proteins, streptavidin-HRP conjugate (Thermo Fisher Scientific) was used to incubating membranes after blocking. Finally, protein bands were visualized by an enhanced chemiluminescence detection system.
9
Western Blot Analysis of Cellular Proteins
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Whole-cell extracts were prepared in cell lysis buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, protease inhibiter [Roche]) with sonication (30 s ON/90 s OFF × 6 times) using Bioruptor (Cosmobio). Protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane using a semi-dry transfer system (Trans-Blot Turbo, BioRad). The transferred membrane was blocked with TBS-T (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween20) containing 5% BSA. The membrane was incubated 1 hour in a primary antibody diluted with Can Get Signal solution 1 (TOYOBO), and washed with TBS-T three times. The membrane was then incubated for one hour in a secondary antibody diluted with Can Get Signal solution 2 (TOYOBO). Washing three times with TBS-T, signals were developed with ECL prime western blotting detection reagents (GE Healthcare), and subsequently detected using the Chemidoc analyzer (BioRad). Primary antibodies were used at the following dilution rate. Anti-actin antibody (Thermo Fisher Scientific, MS-1295-P1): 1/1000. Anti-H3 antibody (Abcam, ab1791): 1/10,000. Anti-GFP antibody (MBL, 598): 1/5000. Anti-rabbit IgG (Promega, W4011) or anti-mouse IgG (Promega, W4021) were used as secondary antibodies at 1/10,000 dilution.
10
Purification and Analysis of PTEN and its Interactions
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Recombinant wild-type PTEN was purified as described previously [30 (link)]. 15s-HpETE and 15s-HETE were purchased from Cayman Chemical (Ann Arbor, MI, USA). NAP-5 Sephadex G25 columns were purchased from GE Healthcare Life Sciences (Little Chalfont, UK). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Lipofectamine 2000 transfection reagent, and anti-actin antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The PTEN and Trx antibodies were prepared as described previously [39 (link), 40 (link)]. Anti-rabbit IgG horseradish peroxidase-conjugated antibody was purchased from Ab Frontier (Daejeon, Korea).
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