Cell lysates from PBMCs were separated by SDS/PAGE gel and transferred to nitrocellulose membranes. Specific proteins were detected using anti-human TfR1 mAb (H68.4, Invitrogen) and blots were re-probed with anti-actin mAb (Chemicon) as a loading control.
Anti actin mab
Manufactured by Merck Group
Sourced in United States
Anti-actin mAb is a monoclonal antibody that binds to actin, a cytoskeletal protein found in eukaryotic cells. The core function of this product is to detect and quantify actin in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti human tfr1 mab h68, by Thermo Fisher Scientific (2 mentions)
Luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab98831, by Abcam (1 mentions)
Casein blocking buffer, by LI COR (1 mentions)
Ibright fl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by Thermo Fisher Scientific (1 mentions)
As11 1630, by Agrisera (1 mentions)
Anti gm130 mab, by BD (1 mentions)
Anti gfp antibody, by BD (1 mentions)
Anti pkm1 2, by Cell Signaling Technology (1 mentions)
Anti g6pd, by Cell Signaling Technology (1 mentions)
Anti hexokinase 2, by Cell Signaling Technology (1 mentions)
Anti hexokinase 1, by Cell Signaling Technology (1 mentions)
Irdye 800, by LI COR (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
0.2 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti smad4, by Santa Cruz Biotechnology (1 mentions)
Anti smad2, by Thermo Fisher Scientific (1 mentions)
Anti smad3, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti actin mab
1
Western Blot Analysis of TfR1
2
Western Blot Analysis of TfR1
3
Cell Lysis and Immunoblot for PrP Detection
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Postnuclear cell lysis, proteinase K (PK) digestion, and immunoblot were done as described [55 (link)]. Briefly, cell cultures were washed with ice cold PBS and then incubated for 10 min with 1 ml ice cold lysis buffer (10 mM Tris-HCL pH 7.5, 100 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate). Supernatants obtained upon 1 min centrifugation at 14,000 rpm were used for PK digestion (20 μg/ml PK; 30 min; 37 °C) or PNGaseF deglycosylation. PK digestion was stopped by addition of Pefabloc protease inhibitor, and then, proteins were precipitated by adding 5 volumes of methanol and overnight incubation at − 20 °C. Protein precipitates were resuspended in TNE buffer (Tris-Cl 10 mM pH 7.5, NaCl 150 mM, EDTA 1 mM), loading buffer was added, samples were boiled for 5 min, and aliquots were subjected to SDS-PAGE and immunoblot as described [55 (link)] using monoclonal antibodies (mAbs) 4H11or 3F4 for detection of PrP; β-actin (loading control) was detected using an anti-actin mAb (Sigma).
4
Myo1g Protein Detection by Western Blot
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For Western blot (WB) analysis, equal amounts of protein (50 µg) in RIPA buffer from each cell type were resolved by 12% SDS-PAGE under reducing conditions. Gels were transferred to nitrocellulose membranes (Bio Rad, Hercules, CA, USA), blocked with 5% non-fat milk, washed with PBS 0.05% Tween 20 (v/v) (PBS-T), and then incubated for 1 h at RT with a 1:1000 dilution of purified rabbit IgG to Myo1g as a positive control and a 1:4000 dilution of supernatant (SN) from the 3 mAb to Myo1g in PBS-T, or a 1:5000 dilution for anti-actin mAb (Sigma-Aldrich, St Louis, MO, USA). Subsequently, they were washed 3 times with PBS-T, and incubated for 1 h at RT with a 1:15,000 dilution of HRP-coupled secondary Ab GAR (Abcam, Cambridge, UK), 1:20,000 of GAM (Abcam, Cambridge, UK). After 3 further washes with PBS-T, luminol reagents were added (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The chemiluminescence analysis was performed on a Fusion FX-7 Spectral Imaging System (Vilber, Paris, France).
5
Protein Expression and Immunoblotting
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Seeds or seedlings were ground directly in 1× or 2× Laemmli loading buffer, respectively, microfuged 10 min at 4 °C to pellet debris, then boiled 5 min prior to fractionation by SDS-PAGE (10% polyacrylamide). Proteins were transferred to nitrocellulose filters, as described in [32 (link)]. Filters were blocked with Casein blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), then co-incubated with anti-GFP mAb (1:10,000, UBPBio, Aurora, CO, USA) and anti-RGA pAb (1:2000, AS11 1630, Agrisera, Vännäs, Sweden) primary antibodies, followed by anti-mouse and anti-rabbit secondary IRDye 800 conjugated IgGs, and visualized using the 800 channel of the Licor Odyssey Infrared Imaging System or the iBright FL1500 Imaging System (Invitrogen, ThermoFisher Scientific, Waltham, MA USA). Filters were subsequently probed with anti-ABI5pAb (1:10,000, Ab98831, AbCam, Cambridge, UK) and anti-actin mAb (A0480, Sigma, St. Louis, MO, USA), followed by anti-mouse secondary IRDye 800 conjugated IgGs (LI-COR Biosciences, Lincoln, NE, USA).
6
Monoclonal Antibodies for HCMV Analysis
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The mouse monoclonal antibodies (MAbs) against viral proteins utilized in this project included antibodies to pp150 (36-14), pp28 (41-18), pp65 (28-19), IE1 (p63-27), gB (27-156), gM (IMP), UL85 (pUL85), UL44 (28-21), and gH (AP86). The commercially available antibodies used in these studies were anti-Grasp65 rabbit polyclonal antibody (Thermo-Fisher, Waltham, MA), anti-GM130 rabbit polyclonal antibody (Thermo-Fisher, Waltham, MA), anti-GM130 MAb (BD Biosciences, San Jose, CA), anti-golgin-245 MAb (BD Biosciences, San Jose, CA), anti-actin MAb (EMD Millipore, Billerica, MA), anti-GFP antibody (BD Biosciences, San Jose, CA), and anti-HCMV UL57 antibody (Virusys, Taneytown, MD).
7
Immunoblotting Analysis of Mitophagy Proteins
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Cell were lysed with RIPA buffer and subjected to SDS-Page and immunoblotting according to standard methods essentially as described [8 (link),19 (link)]. Primary antibodies were anti-MFN2 mAB (1:500; Abnova, Taipei City, Taiwan), anti-G6PD (1:1000; Cell Signaling, Danvers, MA, USA), anti-PKM1/2 (1:1000; Cell Signaling), anti-Hexokinase I (1:1000; Cell Signaling), anti-Hexokinase II (1:1000; Cell Signaling) and anti-Actin mAB (1:4000; Merck Millipore, Burlington, MA, USA). Protein bands were revealed and analyzed following incubation for 1 h at room temperature with a secondary goat anti-mouse IgG antibody conjugated to an infrared fluorescent dye (IRDye 800, Licor, Bad Homburg, Germany) using the Odyssey near infrared laser imaging system (Licor, Bad Homburg, Germany). For mitophagy induction, cells were treated with 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for the indicated time under normal culturing conditions. The reaction was stopped by washing steps with PBS and cell lysis with RIPA buffer.
8
Immunoblotting Analysis of H. capsulatum Proteins
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Proteins from H. capsulatum yeast-like cells, treated or not with mebendazole, were separated by 12% SDS-PAGE [32 (link)], and electrotransferred to 0.2 μm nitrocellulose membranes (Bio-Rad, Feldkirchen, Germany). The membrane strips were blocked at room temperature for 1 h in PBS containing 5% (w/v) non-fat skim milk and supplemented with 0.2% Tween 20 (pH 7.2) (T-PBS). The membranes were then washed three times with T-PBS (5 min per wash). Next, each membrane strip was incubated overnight at 4 °C with antibodies diluted in T-PBS: (a) monoclonal antibody (mAb) anti-beta-tubulin [1/1000 (v/v)] (protein molecular mass 50 kDa) [Sigma Aldrich, St. Louis, MO, USA] or (b) mAb anti-actin [1/1000 (v/v)] (protein molecular mass of 42 kDa) [Sigma Aldrich, St. Louis, MO, USA]. Three 5 min washes with T-PBS followed this step. The membrane strips were incubated as described above with horseradish-peroxidase conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) diluted in T-PBS (1:3000) at 0.16 μg/mL. Following incubation, the membrane strips were washed and developed with SuperSignal West Dura Chemiluminescent substrate (Pierce, Rockford, IL, USA). Lastly, the X-ray films were exposed and developed according to the manufacturer’s instructions (Kodak, Rochester, NY, USA). The experiment was performed twice, on different days.
9
Comprehensive Antibody Immunostaining Protocol
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