RAW264.7 macrophages were seeded in 12-well plates, grown to confluence, and then inoculated with 10 µg/ml or 50 µg/ml purified MBP-TcpF or purified MBP control protein for 5 hours at 37°C. Cells were then washed five times with cold PBS and lysates prepared after treatment with trypsin at a final concentration of 0.25% for 5 minutes at 37°C. Proteins from cell lysate were resolved on a 10% SDS polyacrylamide gel and transferred to PVDF membrane. The membrane was blocked with 5% milk and probed first with anti-MBP monoclonal antibody (New England Biolabs, Ipswich, MA) overnight at 4°C followed by goat anti-mouse IgG conjugated to HRP. Bound antibody was detected using a chemiluminescent detection kit (Thermo Scientific, Rockford, IL).
Monoclonal anti mbp antibody
Manufactured by New England Biolabs
Sourced in Germany
The Monoclonal anti-MBP antibody is a laboratory reagent used to detect and quantify the presence of Maltose Binding Protein (MBP) in biological samples. This antibody is specific to the MBP protein and can be used in various immunoassay techniques, such as Western blotting, ELISA, and immunoprecipitation.
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Lab products found in correlation
Chemiluminescent detection kit, by Thermo Fisher Scientific (1 mentions)
Anti myc monoclonal antibody, by Cell Signaling Technology (1 mentions)
Anti α tubulin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Immobilon fl, by Merck Group (1 mentions)
Perfection v850 pro scanner, by Epson (1 mentions)
Alexa 680 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Glutathione agarose beads, by GE Healthcare (1 mentions)
Anti gfp monoclonal antibody, by Takara Bio (1 mentions)
Anti phospho p44 42 mitogen activated protein kinase, by Cell Signaling Technology (1 mentions)
5 protocols using monoclonal anti mbp antibody
1
MBP-TcpF Protein Internalization in Macrophages
2
Western Blot Analysis of E. coli Proteins
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Total protein of whole E. coli cells (2.5 × 107) or membrane proteins (2 µg) of the membrane homogenates were separated by 10% Bis-Tris SDS-PAGE and transferred to a 0.45 µm nictrocellulose membrane (Sartorius, Göttingen, Germany). The membrane was blocked with 3% BSA in 1x TBS (20 mM Tris, pH 7.4, 150 mM NaCl), 0.1% (v/v) Tween20 for 1 h at room temperature. After washing the membrane (1x TBS, 0.1% (v/v) Tween 20) it was incubated with anti-MBP monoclonal antibody (1:10,000) (New England Biolabs, Frankfurt a. M., Germany), for 1 h at room temperature in 1x TBS, 0.2% (w/v) BSA, 0.1% (v/v) Tween 20. Anti-mouse IgE-alkaline phosphatase antibody (1:20,000) (Sigma-Aldrich, Taufkirchen, Germany) was added and the membrane was incubated further for 1 h at room temperature. The blot was developed using 4.2 mg/mL NBT and 2.1 mg/mL BCIP as a substrate in 100 mM Tris/HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2.
3
Immunoblotting Antibody Detection Protocol
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Primary and secondary antibodies used in the immunoblotting are as follows: monoclonal anti-MBP antibody (New England Biolabs, E8032S, 1:10,000 dilution), monoclonal, horseradish peroxidase (HRP)–conjugated, anti-GST antibody (B-14) (Santa Cruz Biotechnology, sc-138 HRP, 1:1000), monoclonal anti-Myc antibody (Cell Signaling Technology, 71D10, 1:3000 dilution), monoclonal, HRP-conjugated, anti-GFP antibody (Miltenyi Biotec, 130-091-833, 1:3000 dilution), monoclonal anti–α-tubulin antibody (Santa Cruz Biotechnology, sc-5286 HRP, 1:1000), anti-mouse immunoglobulin G (IgG)–peroxidase antibody (Sigma-Aldrich, A9044, 1:5000), and anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, 7074, 1:5000 dilution). Signal detection was performed with the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).
4
Tricine-SDS-PAGE and Western Blotting of MBP Fusions
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Proteins were separated using Tricine-SDS-polyacrylamide (10%) gel electrophoresis under reducing (1× sample buffer containing 10 mM DTT) conditions51 (link); an all blue prestained protein standard was obtained from Bio-Rad. Molecular weights of standard proteins are marked on the side of the gel pictures. For documentation, destained gels were scanned using an Epson Perfection V850 Pro scanner with 1200 d.p.i. resolution. Proteins were electrophoretically transferred to PVDF membrane (Immobilon-FL, Merck Millipore) after SDS-PAGE by semi-dry blotting. MBP fusions were detected with a monoclonal anti-MBP antibody (New England Biolabs, diluted 1:4000) as first antibody, an Alexa 680 goat-anti-mouse antibody (Life Technologies, Thermo Fischer Scientific, 0.8 µg ml−1) as secondary antibody and then using the Odyssey Imaging System (Li-Cor Biosciences).
5
Analyzing BZR1 and MAPK Activation
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