F. graminearum protein extraction was performed, as described previously [41 (link)]. The resulting proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA). GFP tagged proteins were detected with monoclonal anti-GFP (ab32146, Abcam, Cambridge, UK) antibody; the GAPDH level was detected by the monoclonal anti-GAPDH antibody (Huabio M1306-4) for protein loading reference. Each experiment was repeated three times.
Ab32146
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab32146 is a laboratory equipment product. It serves as a tool for performing scientific experiments and analyses in a research setting. The core function of this product is to enable specific laboratory procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
A9044, by Merck Group (4 mentions)
Immobilon p transfer membrane, by Merck Group (3 mentions)
Dapi fluoromount g, by Southern Biotech (2 mentions)
Mab1281 clone 235 1, by Merck Group (2 mentions)
Ab150077, by Abcam (2 mentions)
Ab150115, by Abcam (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Ab125096, by Merck Group (1 mentions)
Anti gfp, by Proteintech (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
A1978, by Merck Group (1 mentions)
Ecl chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (1 mentions)
A9169, by Merck Group (1 mentions)
Ab134175, by Abcam (1 mentions)
Ab32064, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab17868, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Ab137675, by Abcam (1 mentions)
22 protocols using ab32146
1
F. graminearum Protein Extraction and Analysis
2
Immunofluorescence Labeling of Neuronal Cultures
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Cultures were fixed immediately following data acquisition in a solution of 4% paraformaldehyde and 4% sucrose (w/v) in PBS, pH 7.4 at room temperature for 8 minutes. Fixed cultures were then washed three times in Dulbecco’s PBS supplemented with Ca2+ and Mg2+ (DPBS), pH 7.4, prior to permeabilization and blocking in a solution of 0.1% (w/v) gelatin and 0.3% Triton-X-100 (v/v) in PBS, pH 7.4 (GTB) for 12–48 hours at 4C.
For experiments using the sub-frame interpolation algorithm, primary cultures were fixed and stained using primary mouse monoclonal anti-ankyrin G (NeuroMab clone N106/36; 1:500), primary rabbit monoclonal anti-GFP (Abcam ab32146, lot YK011702CS, 1:1000), secondary goat anti-rabbit AlexaFluor 488 conjugated (Abcam ab150077, 1:500), and secondary goat anti-mouse AlexaFluor 647 conjugated (Abcam ab150115, 1:500) antibodies.
For experiments on human iPSC derived neurons, cultures were incubated with primary mouse anti-human nuclear antigen antibody (Millipore MAB1281 clone 235-1,1:500) in GTB overnight at 4 °C, then washed three times in DPBS, and incubated with rabbit anti-GFP AlexaFluor 488 conjugated (polyclonal, Life A21311, 1:300) and secondary antibody donkey anti-mouse AlexaFluor 647 (Life A31571, 1:300) in GTB overnight at 4C. Cultures were washed three times in DPBS prior to mounting in DAPI Fluoromount-G (Southern Biotech).
For experiments using the sub-frame interpolation algorithm, primary cultures were fixed and stained using primary mouse monoclonal anti-ankyrin G (NeuroMab clone N106/36; 1:500), primary rabbit monoclonal anti-GFP (Abcam ab32146, lot YK011702CS, 1:1000), secondary goat anti-rabbit AlexaFluor 488 conjugated (Abcam ab150077, 1:500), and secondary goat anti-mouse AlexaFluor 647 conjugated (Abcam ab150115, 1:500) antibodies.
For experiments on human iPSC derived neurons, cultures were incubated with primary mouse anti-human nuclear antigen antibody (Millipore MAB1281 clone 235-1,1:500) in GTB overnight at 4 °C, then washed three times in DPBS, and incubated with rabbit anti-GFP AlexaFluor 488 conjugated (polyclonal, Life A21311, 1:300) and secondary antibody donkey anti-mouse AlexaFluor 647 (Life A31571, 1:300) in GTB overnight at 4C. Cultures were washed three times in DPBS prior to mounting in DAPI Fluoromount-G (Southern Biotech).
3
Protein Isolation and Immunoblot Analysis
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Protein isolation was performed as previously described (Yun et al., 2015 ). The resulting proteins were separated by 10% SDS polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto an Immobilon‐P transfer membrane (Millipore, Billerica, MA, USA). Monoclonal anti‐GFP (ab32146, Abcam, Cambridge, UK), anti‐mCherry (ab125096, Sigma, St Louis, MO, USA), anti‐RFP (ab65856, Sigma) and anti‐Flag (A9044, Sigma) antibodies were used at a 1:10 000 dilution for immunoblot analyses. The samples were also detected with monoclonal anti‐GAPDH antibody (EM1101, HuaAn Biotechnology Co., Ltd, Hangzhou, Zhejiang, China) as a control.
4
Co-Immunoprecipitation of GFP- and Flag-Fusion Proteins
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The GFP- and 3 × Flag-fusion constructs were verified by DNA sequencing and transformed into pairs into PH-1. Transformants expressing a pair of fusion constructs were confirmed by western blot analysis. For Co-IP assays, fresh mycelia (500 mg) of each strain were finely ground and suspended in 1 ml of extraction buffer containing 1% protease inhibitor. After homogenization with a vortex shaker, the lysates were centrifuged at 10 000 × g for 20 min at 4°C, and the supernatants were incubated with anti-GFP (ChromoTek, Martinsried, Germany) agarose. Proteins eluted from agarose were analyzed by western blotting with mouse monoclonal anti-Flag (A9044, Sigma, St. Louis, MO, USA) and rabbit polyclonal anti-GFP (ab32146, Abcam, Cambridge, UK) antibodies. Samples were also detected with a mouse monoclonal anti-GAPDH antibody (EM1101, HuaAn Biotech. Ltd, Hangzhou, China) as a reference. After inoculation with the secondary antibody, goat polyclonal anti-rabbit IgG-HRP (HA1001, HuaAn Biotech. Ltd., Hangzhou, China), or goat polyclonal anti-mouse IgG-HRP (HA1006, HuaAn Biotech. Ltd., Hangzhou, China), chemiluminescence was detected. All blots were imaged using the Image Quant LAS4000 mini (GE Healthcare, Chicago, IL, USA). The experiment was conducted three times independently.
5
Western Blot Analysis of EGFP Expression
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Cells were lysed with 1% Triton X-100 (Sigma) and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which proteins were transferred to the polyvinylidene difluoride membrane (Millipore). EGFP primary antibody (ab32146; Abcam) and secondary antibody (A9169; Sigma) were used to detect EGFP, while beta-actin primary antibody (A1978; Sigma) and secondary antibody (A9044; Sigma) were used to detect beta-actin as a loading control. An ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) was used to detect blots of proteins.
6
Comprehensive Protein Analysis of Cell Cycle and Apoptosis
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Western blot analysis was performed as described previously (11 (link)). Equal protein loading was monitored using an anti-GAPDH antibody (60004-1-IG; Proteintech, Rosemount, IL, USA). Primary antibodies against cyclin D1 (ab134175; Abcam), cyclin E1 (ab88259; Abcam), CDK2 (ab32146; Abcam), CDK4 (ab137675; Abcam), CDK6 (ab124821; Abcam), caspase 3 (ab17868; Abcam), polyADP-ribose polymerase (PARP) (ab32064; Abcam), Bax (ab32503; Abcam), matrix metalloprotein (MMP)-3, E-cadherin, and N-cadherin (Cell Signaling Technology, Danvers, MA, USA) were used.
7
Visualizing Fungal Toxisome Formation
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To observe toxisome formation, 25 μl mycelia of the FgTri1-GFP labeled strain were added to 25 ml TBI medium. After incubation for 24 h at 28 °C and 150 rpm in the dark, AgNPs were added to each flask to generate final concentrations of 1 μg/ml, 1.88 μg/ml (EC50), and 5.15 μg/ml (EC90) and incubated for another 24 h. Treatments without AgNPs were used as controls. The FgTri1-GFP marker indicated toxisome formation and was observed under a Zeiss LSM780 confocal microscope (Gottingen, Niedersachsen, Germany).
The corresponding protein levels of each sample bearing FgTri1-GFP were detected via western blotting. Briefly, ∼500 mg of freshly prepared mycelia from each sample were finely ground in liquid nitrogen. The powder of each ground sample was suspended in 1 ml of extraction buffer. The lysates were homogenized with a vortex shaker and then centrifuged at 4 °C and 10, 000 g for 20 min. The resulting supernatants were analyzed via western blotting with a monoclonal anti-GFP (ab32146, Abcam, Cambridge, UK, 1:5000 dilution) antibody. Samples detected with a monoclonal anti-GAPDH antibody (EM1101, Hua An Biotech. Ltd., Hangzhou, China, 1:5000 dilution) were used as the reference. Each experiment was conducted in triplicate.
The corresponding protein levels of each sample bearing FgTri1-GFP were detected via western blotting. Briefly, ∼500 mg of freshly prepared mycelia from each sample were finely ground in liquid nitrogen. The powder of each ground sample was suspended in 1 ml of extraction buffer. The lysates were homogenized with a vortex shaker and then centrifuged at 4 °C and 10, 000 g for 20 min. The resulting supernatants were analyzed via western blotting with a monoclonal anti-GFP (ab32146, Abcam, Cambridge, UK, 1:5000 dilution) antibody. Samples detected with a monoclonal anti-GAPDH antibody (EM1101, Hua An Biotech. Ltd., Hangzhou, China, 1:5000 dilution) were used as the reference. Each experiment was conducted in triplicate.
8
Immunoblotting of Synaptic Proteins
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The proteins were transferred to a nitrocellulose membrane (66485, Pall Corporation, USA) using Trans Blot Turbo (1704270, Bio-Rad). Membranes were blocked in 10% non-fat milk in Tris buffered saline with Tween (TBST) at room temperature for 1 h and probed with primary antibodies diluted 1:1000-1:10 000 in 5% milk in TBST at 4 °C overnight. The primary antibodies were as follows: anti-drebrin (GTX12350, GeneTex; ab60933, Abcam), anti-GFP (GTX26673, Genetex; ab32146, Abcam), anti-AChR-α1, α3, α5 (838301, BioLegend), anti-rapsyn (ab156002, Abcam), anti-GAPDH (sc-25778, Santa Cruz) and anti-tubulin (ab18251, Abcam). After washing with TBST, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase [anti-rabbit HRP (111-035-144, Jackson Immuno Research, USA), anti-mouse HRP (7076, Cell Signaling, USA), anti-rat HRP (ADI-SAB-200-J, Enzo Life Sciences), and anti-goat HRP (sc-2020, Santa Cruz)]. Proteins were detected with Femto chemiluminiscent substrate (34095, ThermoFisher Scientific) and developed on X-ray films (771468, Carestream, USA).
9
Autophagy and Phosphorylation Assays
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For macroautophagy and ER-phagy, the free GFP and fusion bands were detected by GFP antibody (GFP 1:10,000; Abcam; ab32146, Shanghai, China) with 12% SDS-PAGE. For the MoAtg8 and the MoAtg8-PE turnover assays, the MoAtg8 and the MoAtg8-PE bands were detected using an Atg8 antibody (1:2000, BML; PM090, Beijing, China) with 13.5% SDS-PAGE. For detecting the phosphorylation level, proteins of the Guy11 and ΔMovps13 mutant strains were extracted by TCA-SDS methods. The Mps1 phosphorylation level was detected using a MAPK antibody (Cell Signaling Technology; 9212S, Danvers, MA, USA).
10
Protein Extraction from Fungal Mycelia
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Mycelia for protein extraction were grown in a liquid medium at 25 °C with 180-rpm rotation. The total protein was extracted from mycelia as described previously [19 (link)]. A monoclonal anti-GFP antibody (ab 32146, Abcam, Cambridge, MA, USA) and an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (EM1101, Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China) were used for immunoblot analyses. These experiments were repeated three times independently.
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