Samples of 40 μg were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted electrophoretically to polyvinylidene fluoride membranes. In brief, the membranes were incubated with primary antibodies, and immunoreactivity was detected using a horseradish-conjugated secondary antibody and visualized using enhanced chemiluminescence. The following primary antibodies were used: Mouse anti-HIF-1α antibody (1:1000, abcam, Burlingame, CA, USA ), rabbit anti-p75NTR monoclonal antibody (1:1000, Cell Signaling and Proteintech), rabbit anti-claudin-5 polyclonal antibody (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), goat anti-ZO-1 polyclonal antibody (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA ), and, rabbit anti-β actin monoclonal antibody (1:500, Sigma Aldrich, St. Louis, MO, USA). For densitometry, data were normalized for the actin expression.
Rabbit anti β actin monoclonal antibody
Manufactured by Merck Group
Sourced in United States
Rabbit anti-β-actin monoclonal antibody is a laboratory reagent used to detect and quantify the presence of the β-actin protein in biological samples. β-actin is a widely expressed cytoskeletal protein that is commonly used as a control for normalizing protein expression data in Western blotting and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti hif 1α antibody, by Abcam (1 mentions)
Rabbit anti claudin 5 polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Dmem high glucose culture medium, by Thermo Fisher Scientific (1 mentions)
Realmastermix sybr green kit, by Tiangen Biotech (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Ripa lysis solution, by Solarbio (1 mentions)
Cdna synthesis kit, by Tiangen Biotech (1 mentions)
Bca protein quantification kit, by Solarbio (1 mentions)
Irisin, by Phoenix Pharmaceuticals (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Rabbit anti lc3 polyclonal antibody, by Abcam (1 mentions)
Rabbit anti caspase3 polyclonal antibody, by Cell Signaling Technology (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using rabbit anti β actin monoclonal antibody
1
Western Blot Analysis of Hypoxia Markers
2
Molecular Mechanisms of Irisin-Induced Macrophage Apoptosis
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Mouse RAW264.7 macrophages were purchased from Shanghai Institute of Biochemistry and Cell Biology (China). Irisin was bought from Phoenix Pharmaceuticals (USA). MTT, oil red O, tunicamycin (TM) and rabbit anti-β-actin monoclonal antibody were obtained from Sigma (USA). Rabbit antibodies against CHOP, Bcl-2, p-PERK and p-eIF2α were provided by Santa Cruz (USA). Rabbit anti-ATF6 polyclonal antibody was purchased from Abcam (USA). Goat anti-rabbit IgG and ready-to-use SABC-Cy3 immunohistochemical kit were bought from Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd. (China) and Wuhan Boster Biological Technology Co., Ltd. (China) respectively. DMEM high-glucose culture medium and fetal bovine serum were provided by Gibco (USA). RIPA lysis solution and BCA protein quantification kit were purchased from Solarbio (USA). Annexin V-FITC apoptosis detection kit was bought from Nanjing Keygen Biotech. Co., Ltd. (China). Trizol reagent was obtained from Invitrogen (USA). cDNA synthesis kit and Real Master Mix (SYBR Green) kit were provided by Tiangen Biotech (Beijing) Co., Ltd. (China). ECL kit and PVDF membrane were purchased from Pierce (USA) and Millpore (USA) respectively. Other reagents were all analytically pure.
3
Erythroid Cell Protein Analysis
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CD45–CD71+ bone marrow erythroid cells were lysed and sonicated in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% SDS and 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma-Aldrich). A total of 50 μg protein of the lysates was separated through 15% denaturing polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories). Primary antibodies used were a rabbit anti-caspase 3 polyclonal antibody (Cell Signaling Technology, Danvers, MA), a rabbit anti-LC3 polyclonal antibody (Abcam, Cambridge, UK) and a rabbit anti-β-actin monoclonal antibody (Sigma-Aldrich). Secondary antibody was used was a horseradish peroxidase conjugated goat anti-rabbit IgG (Sigma-Aldrich). The densitometric analysis was performed using ImageJ software (The National Institutes of Health, MD).
4
Examining Survivin Regulation via miRNA
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The rabbit anti-survivin antibody and rabbit anti-β-actin monoclonal antibody were purchased from Sigma (St. Louis, MO, USA); the TaqMan microRNA reverse transcription kit was purchased from invitrogen (Carlsbad, CA, USA).; PVDF membrane was purchased from Shanghai Baoman Biotechnology Co. Ltd. (Shanghai, China); the HRP marked goat anti rabbit IgG was purchased from Sigma (St. Louis, MO, USA); SW579 cells were purchased from Bio-Rad (Beijing, China); siRNA knockdown and control fragments were purchased from Amresco (Solon, OH, USA).
5
Western Blot Analysis for Protein Detection
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Protein preparation was performed as described previously (Mandhan et al., 2006a (link)): the cloaca/hindgut per condition were pooled and sonicated in ddH2O containing protease inhibitors. Protein extracts were seperated on SDS-PAGE electrophoresis, and transferred to PVDF membranes, blocked with 5% fat-free milk in Tris-buffered saline (2 h, room temperature). Membrane were incubated in primary antibody against Wif1 (diluted 1:500, Rabbit polyclonal, ab186845; Abcam, Cambridge, UK), β-catenin (diluted 1:1,500, mouse monoclonal, cat#610154; BD Biosciences, San Jose, CA, USA) or anti- β-Actin rabbit monoclonal antibody (1:2,000 dilution; Sigma, St Louis, MO, USA), and incubated with the secondary antibody (diluted 1:3,000, goat anti-rabbit or goat anti-mouse HRP conjugate; Jackson Immunoresearch, West Grove, PA, USA) Membranes were developed by using a chemiluminescent substrate kit (Pierce, Pierce, Rockford, IL, USA) and densitometric values were analyzed by using the ECL Plus detection system (Millipore, Billerica, MA, USA) (https://www.protocols.io/view/western-blot-analysis-kumcwu6 ).
6
Western Blot Analysis of PLAC1 Protein
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Separated mononuclear cells (1×106) were lysed in 50 μl sample buffer containing 150 mM Tris Ph=6.8, 1.2% SDS, 200 mM 2-ME, 0.2% bromophenol blue and 33% glycerol on ice. The lysates were boiled for 10 min and then centrifuged at 17700×g for 20 min at 4°C. Thirty μl of cell lysates and 35 ng recombinant human PLAC1 (rhPLAC1) (produced in Dr. Zarnani's lab) were run on a 14% SDS-PAGE gel at 100 V for 2 hrs and then transferred to PVDF membrane at 200 mA for one hr. The existence of protein bands was visualized by Ponceau S staining. The membrane was blocked with PBS-0.1% Tween 20 (PBS-T) containing 5% skimmed milk solution overnight without shaking at 4°C. The membrane was then immunoprobed with rabbit anti-rhPLAC1 antibody (produced in Dr. Zarnani's lab) at 2 μg/ml in PBS-T containing 1% skimmed milk solution for 1.5 hr with shaking at RT followed by goat anti-rabbit IgG-HRP (1:6000) (Bio-Rad, Hercules, CA, USA) in PBS-T containing 1% skimmed milk solution for one hr with shaking at RT. After extensive washing, membrane was developed with ECL. Anti-β-actin rabbit monoclonal antibody (Sigma, St. Louis, USA) was used for visualization of β-actin after reprobing of the membrane.
7
Comprehensive Western Blotting Analysis
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Western blotting analysis was performed according to a standard method previously described by using the following antibodies: anti‐MCM5 (Proteintech, Chicago, IL, USA),[52 , 53 ] and anti‐IGF2BP3 (Abcam, Cambridge, UK), anti‐Flag and anti‐HA (Cell Signaling, Danvers, USA), anti‐ cleaved‐Notch1, and anti‐SIRT1 (Immunoway, plano, TX, USA), respectively. Blotted membranes were stripped and re‐blotted with an anti‐p84 rabbit monoclonal antibody (Sigma, St. Louis, MO, USA) or anti‐β‐actin rabbit monoclonal antibody (Sigma, Saint Louis, MO, USA) as loading controls.
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