The commercial antibodies used in this study were as follows: primary antibody anti-β-actin (CWBIO, CW0264M) was used to detect the internal control β-actin; anti-mouse IgG (KPL, 074-1806) and goat anti-mouse IgA (Sigma, A4789) secondary antibodies were used to detect serum IgG and mucosal IgA. The anti-VP6 polyclonal antibody was prepared in a previous study (Dong et al., 2005 (link)). The anti-LTB-NSP4 polyclonal antibody was prepared from rabbit serum immunized with purified HIS-LTB-NSP4 protein by Beijing Protein Innovation Co., Ltd.
Cw0264m
Manufactured by CWBIO
Sourced in United States
The CW0264M is a laboratory equipment designed for scientific research applications. It serves as a tool for conducting various experiments and analyses. The core function of this product is to provide a stable and controlled environment for research activities.
Automatically generated - may contain errors
Lab products found in correlation
Ab62341, by Abcam (2 mentions)
Sra20001, by Agdia (2 mentions)
Anti β actin, by CWBIO (1 mentions)
Goat anti mouse iga, by Merck Group (1 mentions)
Cw0102, by CWBIO (1 mentions)
Cw0103, by CWBIO (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ht101 01, by Transgene (1 mentions)
A5598, by Merck Group (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
7 protocols using cw0264m
1
Antibody Detection for Rotavirus Study
2
Immunoblotting Protein Expression Analysis
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Total proteins were extracted with extraction buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 10 mM MgCl2, 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride, and 1× complete protease inhibitor cocktail [Roche, 04693159001]) and quantified using the Bradford assay (Bio-Rad). Samples were boiled at 95°C for 5 min in 2× SDS loading buffer and separated on SDS–PAGE gels. Proteins were transferred to polyvinylidene difluoride membranes and immunoblotted with an anti-SEU (Huai et al., 2018 (link)) or anti-GFP antibody (TransGen, HT801), with an anti-actin antibody (CWBIO, CW0264M) as a loading control, and then immunoblotted with horseradish peroxidase-conjugated goat anti-rabbit IgG (CWBIO, CW0103S) or goat anti-mouse IgG (CWBIO, CW0102S). Signals were captured with a chemiluminescence imaging system (Biostep).
3
Transient Expression of TaPIF4 and TaSG-D1 in Nicotiana benthamiana
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The constructs 35 S: TaPIF4-Myc, 35 S: TaPIF4A-Myc, 35 S: TaPIF4D-Myc and 35 S: TaSG-D1-GFP/35 S: TaSG-D1E286K-GFP were transformed into Agrobacterium tumefaciens strain GV3101 separately. Various blends of plasmids were co-expressed into 4-week-old N. benthamiana leaves. Between 48 and 96 h after transfection, the total proteins were extracted for immunoblotting. For the TaPIF4 and TaSG-D1/TaSG-D1E286K stability in different temperature gradients, the infiltrated N. benthamiana leaves were treated under 42 °C for 0 h, 0.5 h, 1 h, 3 h. Next, total proteins were extracted for immunoblotting. 10 µL sample was loaded for both immunoblot. The proteins were detected using anti-Myc antibody (1:5000; TransGen Biotech, HT101-01, P21018), anti-GFP antibody (1:5000; TransGen Biotech, HT801-01, O21209), anti β-Actin Mouse Monoclonal antibody (1:5000 dilution; CWBIO, Catalog # CW0264M, Lot # 01265/35721, Clone # 6D1) and the secondary antibody of Goat Anti-Mouse IgG (H&L)-HRP Conjugated (1:5000, EASYBIO, BE0102-100). The primers used for plasmid construction are listed in Supplementary Data 6 .
4
Western Blot Analysis of PVY CP in N. benthamiana
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For western blotting, protein was isolated from N. benthamiana, and total plant proteins were 1:1 equal volume mixed with 2 × SDS-PAGE buffer. Next, the protein samples were incubated at 95 °C for three min and separated on a 12% SDS-polyacrylamide gel. The separated proteins were then transferred onto nitrocellulose membranes by electroblotting instrument. The PVY CP antibody (SRA20001, Agdia, USA), anti-RFP (ab62341, Abcam, Shanghai) and β-Actin (CW0264M, CWBIO, Beijing) antibody were used for this assay.
5
Immunoprecipitation of Zebrafish Gamma-Tubulin
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For immunoprecipitation, HEK293T cells were co-transfected with GFP-tagged zebrafish γ-tubulin and Myc-tagged zebrafish Tubgcp3 constructs using Lipofectamine 2000 Transfection Reagent (Invitrogen). After 48 h, transfected cells were lysed with lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.5% NP-40, protease inhibitor cocktail (04693132001, Roche)) and incubated on ice for 10 min. The lysate was obtained by centrifugation at 16,000 × g for 15 min at 4°C. The supernatants were incubated with anti-Myc antibody and protein A-Sepharose (GE Healthcare) at 4°C for 4 h. Then the precipitants were washed three times with lysis buffer before being boiled in SDS loading buffer for immunoblot. Zebrafish embryos (5 dpf) were collected, washed and homogenized in RIPA lysis buffer (P0013B, Beyotime Biotechnology, China) containing protease inhibitor cocktail (04693132001, Roche). The following primary antibodies were used: rabbit anti-Myc (A5598, Sigma-Aldrich; 1:1000), rabbit anti-GFP (50430-2-AP, ProteinTech; 1:2000), rabbit anti-TUBGCP3 (15719-1-AP, ProteinTech; 1:1000), mouse anti-β-Actin (CW0264M, CWBIO; 1:2000) and rabbit anti-β-Actin (GTX124388, GeneTex; 1:5000).
6
Co-Immunoprecipitation Assay in N. benthamiana
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For Co-IP assay, the ORFs were cloned into pCAMBIAI1300-GFP and pCAMBIAI1300-3×FLAG vectors (Supplemental Figures 13B and15A), respectively. The positive plasmids were transformed into A. tumefaciens strain EHA105 and infiltrated into N. benthamiana leaves. The inoculated plants were kept in dark for 24 h and cultured in light for 24 h. The total proteins of inoculated leaves were extracted with lysis buffer with freshly added phenylmethylsulfonyl fluoride (10 mM). Extracts were then clarified by centrifugation at 4°C for 20 min. Immunoblots were performed as described (Pedmale and Liscum, 2007) . Anti-GFP (1:2000; Roche, 11814460001), anti-FLAG (MBL, M185-7), and anti-mouse IgG (1:75000, Sigma, A9044-2ML) were used to detect GFP-and FLAG-tagged proteins accordingly. Actin (1:5000, CWBIO, CW0264M) was used as a loading control.
7
Western Blot Analysis of PVY CP and RFP
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For western blotting, protein was isolated from N. benthamiana, and total plant proteins were 1:1 equal volume mixed with 2×SDS-PAGE buffer. Next, the protein samples were incubated at 95°C for three min and separated on a 12% SDS-polyacrylamide gel. The separated proteins were then transferred onto nitrocellulose membranes by electroblotting instrument. The PVY CP antibody (SRA20001, Agdia, USA), anti-RFP (ab62341, Abcam, Shanghai) and β-Actin (CW0264M, CWBIO, Beijing) antibody were used for this assay.
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