Pulldown assays were performed in buffer containing 10 mM HEPES (pH 7.0), 150 mM NaCl, 5% Dimethyl sulfoxide (DMSO), and 5 mM 2-mercaptoethanol at 25 °C. MBP-His tagged eVP35 IID WT protein was immobilized on amylose resin, incubated with purified His-tagged NP protein, and subsequently washed. For small molecule competition assays, 500 μM small molecules was incubated with MBP-His tagged eVP35 IID prior to incubation with His-tagged NP. Following washout of unbound material, beads containing MBP-His tagged eVP35 IID WT protein (or His tagged eVP35 IID/His-tagged NP complex) was resolved on SDS-PAGE and Western blotted with mouse anti-His antibody (Santa Cruz biotechnology), followed by horseradish peroxidase (HRP) conjugated goat anti-mouse antibody (Bio-Rad). Membranes were developed using Millipore Immobilon Western Chemiluminescence HRP substrate and recorded on a ChemiDoc (Bio-Rad).
Mouse anti his antibody
Manufactured by Santa Cruz Biotechnology
The Mouse anti-His antibody is a primary antibody that recognizes the polyhistidine (His) tag. It is commonly used for the detection and purification of recombinant proteins expressed with a His-tag.
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Lab products found in correlation
Horseradish peroxidase hrp conjugated goat anti mouse antibody, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Mouse anti flag antibody, by Merck Group (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Mouse anti ub antibody, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
4 protocols using mouse anti his antibody
1
eVP35 IID-NP Protein Interaction Assay
2
Western Blot Analysis of Microbial Proteins
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Protein samples were separated on 10% or 15% gels by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained with 0.25% Coomassie Brilliant Blue R250 (Sigma-Aldrich; 20,278) reagent or transferred to 0.45 μm polyvinylidene fluoride membranes (Pall; BSP0861) for immuno-blotting analysis. The membranes were incubated at 4°C overnight with one of the following primary antibodies: mouse-anti-His antibody (1:200; Santa Cruz biotechnology; sc-8036), mouse-anti-Lewis Y monoclonal antibody (1:1000, Santa Cruz Biotechnology; sc-59,472), rabbit-anti-CagA monoclonal antibody (1:2000; Santa Cruz Biotechnology; sc-25,766), goat-anti-UreA monoclonal antibody (1:3000; Santa Cruz Biotechnology; sc-21,016), and visualized by LI-Cor Odyssey® Infrared Imaging System.
3
Dlg-Gukholder Protein Binding Assay
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GST fusions of Dlg fragments and mutants were incubated with glutathione agarose beads at 4°C for 30 min and subsequently washed three times with binding buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 0.002% Triton X-100). His-tagged Gukholder (amino acids 749–1044) was added to washed beads and incubated at 4°C for 1 hr. The reactions were then washed three times with binding buffer, eluted by addition of SDS loading buffer, and ran on a 12.5% SDS polyacrylamide gel. SDS-PAGE gels were transferred onto nitrocellulose and blocked in 5% skim milk in TBS-T. Blots were subjected to Western analysis by probing with a primary mouse anti-His antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz CA) and IRDye 800CW goat anti-mouse secondary antibodies and visualized using LiCOR Odyssey.
4
Quantitative Western Blot Analysis of Cardiac Proteins
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Western blot was conducted to test the protein levels in mouse ventricular tissue and neonatal cardiomyocytes. We take the 2 mm area around the infarct of the pathological mouse heart as the peri-infarcted region. After quantification with the BCA Protein Assay kit (Beyotime, Shanghai, China), an equal amount of 60 μg protein from each group was fractionated by 8% SDS-PAGE and electro-blotted onto NC membranes (Pall, New York, USA). Membranes were incubated with primary antibodies overnight at 4 °C, including rabbit anti-Meis1 antibody (Abcam, UK, Cat#: ab19867, 1:1000), rabbit anti-Nav1.5 antibody (Alomane laboratory, Israel, Cat#: ASC-005, 1:200), mouse anti-CDC20 antibody (Proteintech Group, America, Cat#: 10252-1-AP, 1:500), mouse anti-Ub antibody (Santa Cruz Biotechnology, Europe, Cat#: sc-8017, 1:1000), mouse anti-His antibody (Santa Cruz Biotechnology, Europe, Cat#: sc-57598, 1:200) and mouse anti-flag antibody (Sigma, USA, Cat#: F1804, 1:1000). Mouse anti-β-Actin antibody (Zhongshanjinqiao, Beijing, China, Cat#: TA-09, 1:1000) was used as an internal reference. The Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebraska, USA) was used for image capture and Image studio software was used to quantify bands in each group.
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