To detect the CEP70 protein, testes of Cep70−/− and WT male mice were pulverized in liquid nitrogen and collected in 500 μL ice-cold Radio Immunoprecipitation Assay Lysis Buffer (RIPA) buffer containing protease inhibitors. After that, the lysates were diluted with 2× Laemmli sample buffer (Bio-Rad, 1610737), and boiled in water for 10 min. The protein samples were separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE, 10% acrylamide running gel) and then electrically transferred to polyvinylidene fluoride (PVDF) membranes. After transfer, the PVDF membranes were blocked with 5% skimmed milk in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.5) containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then immunoblotted at 4 °C overnight with Anti-CEP70 antibody (Abcam, ab227456, 1:1000, Rabbit) and Anti-β-actin mouse monoclonal antibody (TransGen Biotech, HC201, 1:1000). After washing in TBST, the membranes were incubated for 1 h with Anti-rabbit or mouse HRP-conjugated secondary antibody (1:3000). Finally, protein bands were visualized by an enhanced chemiluminescence detection system (Tanon-5200). Western blot images were processed using ImageJ software (Wayne Rasband, USA).
Mouse anti β actin monoclonal antibody
Manufactured by Transgene
Sourced in China
Mouse anti-β-actin monoclonal antibody is a laboratory reagent used to detect and quantify the presence of the β-actin protein in biological samples. It is a highly specific antibody that binds to the β-actin isoform, a ubiquitous cytoskeletal protein found in all eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated rabbit anti mouse igg, by Cell Signaling Technology (2 mentions)
Ab227456, by Abcam (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Hc201, by Transgene (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Hs201 01, by Transgene (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Microchemi 4, by DNR Bio-Imaging Systems (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Proteintech (1 mentions)
Alexa 647 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Goat anti mouse igg hrp, by Transgene (1 mentions)
Anti gapdh, by Transgene (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Ru486, by Merck Group (1 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse igg, by Transgene (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
9 protocols using mouse anti β actin monoclonal antibody
1
Western Blot Analysis of CEP70 in Mouse Testes
2
Worm Protein Extraction and Western Blot
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Worm lysates were prepared using lysis buffer in the presence of protease inhibitors (Sigma-Aldrich, USA) or protease and phosphatase inhibitors (Sigma-Aldrich, USA). Proteins were boiled for 10 min at 98°C before gel electrophoresis. For the western blotting, we used primary anti-phospho-p38 MAPK rabbit polyclonal antibody (Cell Signaling Technology, 4511, USA) at a 1:1,000 dilution, or anti-β-actin mouse monoclonal antibody (Transgen, HC201-01, China) at a 1:3,000 dilution. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Transgen, HS101-01, China), or anti-mouse (Transgen, HS201-01, China) were used as secondary antibody at a 1:2,000 or 1:5,000 dilution, respectively. Images of western blot membranes were captured using an imaging system (MicroChemi 4.2, DNR Bio Imaging Systems). All western blotting experiments were repeated at least three times independently.
3
Visualizing STAT1 in Living Cells
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Mammalian expression plasmids encoding wild-type human STAT1 fused carboxy-terminally to green fluorescent protein (a gift from Prof. Yue Qin) were used for living cell fluorescence imaging. Alexa647 goat anti-mouse IgG (Invitrogen, A-21236) was used as a non-specific STAT1 antibody. Western blot analyses were done with the following primary antibodies: anti-STAT1α p91 (a mouse monoclonal antibody epitope mapping between amino acids 613–739 of STAT1α p91 of human origin, from Santa Cruz, C-111) and anti-β-Actin mouse monoclonal antibody (Transgen Biotech, #I10813). Peroxidase-conjugated goat anti-mouse IgG (Proteintech, SA00001-1) was used as the secondary antibody.
4
HSV-1 Infection Regulatory Pathway Analysis
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CORT (Sigma, 27840), RU-486 (Mifepristone, Sigma, M8046), 3-MA (M9281), cycloheximide (CHX, C4859) and BaF1 (B1793) were purchased from Sigma. Rapamycin (S1039) and MG132 (S2619) were purchased from Selleck. The following antibodies: anti-HSV-1 + HSV-2 gB (10B7) (Abcam, ab6506), anti-HSV-1+HSV-2 ICP27 antibody(1-L-11) (Abcam, ab31631), anti-HSV-1 ICP8 major DNA binding protein antibody (11E2) (Abcam, ab20194), anti-ICP0 antibody (Abcam, ab6513), anti-PML (Abcam, ab96051), anti-PML (Proteintech, 21041-1-AP), anti-ULK1 (Sigma, A7481), anti-ATG5 (C-terminal) (Sigma, A0731), anti-LC3B antibody (Sigma, L7543), anti-LC3B antibody (CST, 2775), anti-P62 (Abcam, ab56416), anti-Beclin-1 (Santa Cruz, sc-11427), anti-HA (Abcam, ab9110), anti-TMEM173 (anti-STING, Proteintech, 19851-1-AP), anti-MAVS (Proteintech, 14341-1-AP), anti-IFNβ (Abcam, ab85803), anti-cGAS (Abcam, ab176177), anti-phospho-TBK1 (CST, 5483), anti-TBK1 (CST, 3504), anti-GAPDH (Transgen, HC301), anti-β-Actin mouse monoclonal antibody (Transgen, HC201), goat anti-mouse IgG-HRP (FUDE, FDM007) and goat anti-rabbit IgG-HRP (FUDE, FDR007) were used for immunoblot analysis.
5
Western Blot Analysis of His-tagged Proteins
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DEF cells were lysed with RIPA buffer (beyotime Biotech, Shanghai, China) at 36 h post transfection. Cell lysates were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the isolated proteins were electroblotted on PVDF membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 5% milk for 1.5 h in TBST (0.1% Tween-20 in PBS) and then incubated with mouse anti-His monoclonal antibody (Ruiyingbio, Suzhou, China) or mouse anti-β-actin monoclonal antibody (Transgen Biotech, Beijing, China) for 2 h, in 37℃. HRP-conjugated goat anti-mouse IgG (Transgen Biotech) antibody 1:5000 was used as the secondary antibody, incubate in 37℃ for 1h. The proteins were visualized by chemiluminescence using an ECL kit (Bio-Rad, USA).
6
Mapping MAPK Signaling in Brain Endothelial Cells
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Human brain microvascular endothelial cells and MECs were collected after 48 h of infection. The total protein was extracted from both cell types and lysed in Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Hayman, China). The cell extract was centrifuged at 12,500 × g at 4°C for 25 min. Thereafter, the total protein (60 μg) was fractionated on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes for western blotting. Western blotting was performed by using a specific Phospho-MAPK Family antibody sampler kit (#9910) from Cell Signaling Technology (Danvers, MA, United States) to detect MAPK signaling pathway-related proteins phospho (p)-ERK (extracellular regulated kinase), p-JNK (JUN N-terminal kinase) and p-p38. Primary antibodies against the TNF-α (#6945) was also purchased from Cell Signaling Technology Inc. The primary antibodies against ERβ (#ab3576) and TLR4 (#ab13556) were purchased from Abcam company. β-actin was detected as an internal control using a mouse anti-β-actin monoclonal antibody (#HC201-02, TransGen Biotech, Beijing, China). The secondary antibodies against mouse IgG (#7076) and rabbit IgG (#7074) were purchased from Cell Signaling Technology Inc.
7
Western Blot Analysis of Protein Expression
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Cells were cultured in 12-well plates and harvested with lysis buffer containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific). Equal amounts of the samples were then separated by SDS-PAGE using 10% polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes, incubated with appropriate primary and secondary antibodies, and visualized using an enhanced chemiluminescence system (Bio-Rad). The expression of β-actin, which was used as a loading control, was detected with mouse anti-β-actin monoclonal antibody (Transgen Biotech).
8
Western Blot Analysis of Ptd1 and PTD1
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Total proteins extracted from ground stems of WT and Ptd1 plants were separated by 10% SDS-PAGE (for β-actin protein, MW 42 kDa) or 16% Tris-Tricine SDS-PAGE. Following electrophoretic separation, the protein samples were transferred to polyvinylidene difluoride (PVDF) membranes. After overnight blocking, the membranes were incubated with a solution of primary antibodies diluted in phosphate-buffered saline (1/10 000). Finally, the membranes were stained with a secondary antibody and the bands were detected using a chemiluminescence imaging system (Tanon 5200, Shanghai, China) using enhanced chemiluminescence (ECL, Amersham, USA). The 1–100th aa sequence shared by Ptd1 and PTD1 was used as the antigen to produce an anti-PTD1/Ptd1 polyclonal antibody in rabbits (Beijing Genomics Institute, Shenzhen, China). The mouse anti-β-actin monoclonal antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) or anti-mouse IgG (H+L) secondary antibodies (Transgen Biotech Co., Beijing, China) were used in the assays. Ptd1 and PTD1 are expected to have an N-terminus 27-aa extracellular signal peptide, so the molecular weights of the mature proteins (of which the signal peptide is excised) should be 9. 1 kDa and 9. 46 kDa, respectively.
9
Antibody Generation and Reagent Procurement
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The rabbit anti-NP polyclonal antibody and mouse anti-M1 monoclonal antibody were generated as previously described [71 (link)]. The rabbit anti-NS1 polyclonal antibody was generated as previously described [72 (link)]. The mouse anti-FLAG M2 monoclonal antibody (Sigma-Aldrich, USA), mouse anti-Myc monoclonal antibody (Santa Cruz Biotechnology, USA), mouse anti-His monoclonal antibody (Beyotime, China), rabbit anti-IAV PB1 polyclonal antibody (GeneTex, USA), rabbit anti-IAV PB2 polyclonal antibody (GeneTex, USA), mouse anti-Lamin B1 monoclonal antibody (Santa Cruz Biotechnology, USA), mouse anti-β-actin monoclonal antibody (TransGen Biotech, China), mouse anti-GAPDH monoclonal antibody (Abcam, UK), HRP-conjugated goat anti-mouse IgG (Beyotime, China), HRP-conjugated goat anti-rabbit IgG (Beyotime, China), HRP-conjugated rabbit anti-mouse IgG (Cell Signaling Technology, USA), and HRP-conjugated mouse anti-rabbit IgG (Cell Signaling Technology, USA) were purchased from the indicated companies. Poly(I:C) sodium salt (Sigma, USA), human IFN-β (PeproTech, USA), RNase R (Lucigen, USA), RiboLock RNase Inhibitor (Thermo Fisher Scientific, USA), protease inhibitor cocktail (Roche, Switzerland), isopropyl-β-D-thiogalactopyranoside (IPTG; Calbiochem, USA), and tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich, USA) were purchased from the indicated companies.
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