PIP strip membranes (Life Technologies) were blocked in blocking buffer (3% fat-free bovine serum albumin [Sigma] in TBS [20 mM Tris-HCl, pH 7.5, 500 mM NaCl]) for 1 h at room temperature with mild agitation. Blocked PIP strips were then incubated in 0.4 µM MBP-Stv1NT-FLAG in blocking buffer for 1 h. Bound protein was detected by incubation with mouse monoclonal anti-FLAG M2 antibody (Sigma) followed by horseradish peroxide–conjugated anti-mouse antibody (Bio-Rad). All incubations were for 1 h at room temperature and were followed by three TBS washes for 10 min. Finally, the strip was treated with a chemiluminescent substrate (Thermo Scientific) for 5 min and developed by exposure to film for 5–20 s. The control MBP strip was probed with mouse monoclonal anti-MBP antibody (NEB).
Mouse monoclonal anti mbp antibody
Manufactured by New England Biolabs
The Mouse monoclonal anti-MBP antibody is a laboratory reagent used for the detection and identification of the Myelin Basic Protein (MBP) in various sample types. This antibody is produced in mice and is specific for the MBP protein.
Automatically generated - may contain errors
Lab products found in correlation
Pip strip membranes, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Mouse monoclonal anti polyhistidine antibody, by Merck Group (1 mentions)
Amersham protran, by Cytiva (1 mentions)
Power blotter apparatus, by Thermo Fisher Scientific (1 mentions)
Instantblue, by Abcam (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Rapid pngase f, by New England Biolabs (1 mentions)
Envicarb 100 mg, by Merck Group (1 mentions)
Millicell ez slide, by Merck Group (1 mentions)
Alexa fluor 594 conjugated goat anti mouse immunoglobulin g igg, by Thermo Fisher Scientific (1 mentions)
Carv 2 confocal microscope, by Zeiss (1 mentions)
Tev protease, by New England Biolabs (1 mentions)
Prism 8, by GraphPad (1 mentions)
Rabbit monoclonal anti ha tag, by Cell Signaling Technology (1 mentions)
7 protocols using mouse monoclonal anti mbp antibody
1
Phosphoinositide Binding Protein Assay
2
Western Blotting of Polyhistidine-Tagged Proteins
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Proteins were loaded on 12% polyacrylamide gel, with color Prestained Protein Standard (NEB). After migration, proteins were stained with Instant Blue (Abcam). For Western blotting, proteins were transferred onto a nitrocellulose membrane (Amersham Protran, Cytiva) using a Power Blotter apparatus (Invitrogen). Membranes were then saturated in PBS containing 0.1% Tween 20 (PBST) and 4 % skim milk overnight. They were incubated for 2 h with PBST containing a 1:2000 dilution of mouse monoclonal anti-polyHistidine antibody (Sigma-Aldrich) or alternatively with mouse monoclonal anti-MBP antibody (NEB), and then with PBST containing a 1:1000 dilution of Goat anti-mouse IgG Peroxydase conjugated (Invitrogen) for 1 h. Immunodetected proteins were revealed with Pierce ECL Western Blotting Substrate (Thermo), following the manufacturer’s instructions, and chemiluminescence was detected with a ChemiDoc XRS+ system (Bio-Rad).
3
Deglycosylation of Antibodies for Lectin ELISA
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Aliquots (150 μg) of mouse monoclonal anti-MBP antibody (New England Biolabs), human or rabbit polyclonal IgG (Sigma-Aldrich) were incubated with Rapid PNGase F (New England Biolabs) in a total volume of 50 μL of Rapid PNGase F reaction buffer for 10 min at 50 °C. For use in lectin ELISA experiments, the deglycosylated antibodies were dialyzed against 20 mM sodium phosphate, pH 7.4, 150 mM NaCl using 10,000 Da MWCO dialysis membranes. For glycan labeling, the liberated N-glycans were cleaned and desalted using SPE Supelclean columns ENVI-18 100 mg and ENVI-CARB 100 mg (Supelco).
4
Fluorescent Imaging of Protein Internalization
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U251, U251+PTEN or Astrocyte cells were treated with purified 2XMBP-scFv proteins for 24 hours in chamber well slides (Millipore Millicell EZ Slides). Cells were fixed with 1% paraformaldehyde/2% sucrose for 15 minutes at room temperature, followed by 100% methanol for 30 minutes at -20°C, and 50% methanol/50% acetone for 20 minutes at -20°C. Slides were then incubated in permeabilization/blocking solution (10% BGS, 0.5% Triton X-100 in phosphate-buffered saline (PBS)) at room temperature for 1 h. Primary antibody (mouse anti-MBP monoclonal antibody, New England BioLabs, Inc. #E8032S) was diluted 1:500 in permeabilization/blocking solution and used to stain cells at 4°C overnight. The secondary antibody used was Alexa Fluor 594-conjugated goat anti-mouse immunoglobulin G (IgG) (Life Technologies). Three washes with PBS with Triton X-100 and four washes with PBS were each performed after primary incubation and after secondary incubation. Cells were costained with DAPI to visualize the nuclei. Slides were imaged on a Zeiss-CARV II confocal microscope.
5
In Vitro TEV Digestion of MBP-SAG1 Fusion
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10% or 12% SDS-PAGE, silver staining and Western blotting were performed using standard protocols.
Staining/destaining of nitrocellulose membranes with DirectBlue 71 was performed as described [68] .
The following primary and secondary antibodies were used with the indicated dilutions: mouse anti-MBP monoclonal antibody (NEB) ( [71] in conjunction with packages drLumi [72] and pROC [73] [69]. For other analyses Prism 8 (GraphPad) was used.
In vitro TEV digestion and analysis of MBP tev -SAG1 bio -His 6 F
For in vitro TEV digestion, 1.5 µg puri ed MBP tev -SAG1 bio -His 6 were rst incubated either without or with 10U TEV protease (NEB) in a nal volume of 50 µl 1x TEV reaction buffer and incubated at 30°C for 1h or 4h, or overnight at 4°C. After incubation, 3x10 4 Sav-coated MagPlex® beads were added, incubated with shaking for 1h, then beads were washed and resuspended in PBS/1%BSA. A total of 1,500 beads for each of the different conditions were then analyzed as above, using either the anti-MBP antibody followed by donkey anti-mouse-PE, or human sera followed by anti-human IgG (Fc)-RPE, as described above.
Staining/destaining of nitrocellulose membranes with DirectBlue 71 was performed as described [68] .
The following primary and secondary antibodies were used with the indicated dilutions: mouse anti-MBP monoclonal antibody (NEB) ( [71] in conjunction with packages drLumi [72] and pROC [73] [69]. For other analyses Prism 8 (GraphPad) was used.
In vitro TEV digestion and analysis of MBP tev -SAG1 bio -His 6 F
For in vitro TEV digestion, 1.5 µg puri ed MBP tev -SAG1 bio -His 6 were rst incubated either without or with 10U TEV protease (NEB) in a nal volume of 50 µl 1x TEV reaction buffer and incubated at 30°C for 1h or 4h, or overnight at 4°C. After incubation, 3x10 4 Sav-coated MagPlex® beads were added, incubated with shaking for 1h, then beads were washed and resuspended in PBS/1%BSA. A total of 1,500 beads for each of the different conditions were then analyzed as above, using either the anti-MBP antibody followed by donkey anti-mouse-PE, or human sera followed by anti-human IgG (Fc)-RPE, as described above.
6
Western Blot and Proximity Ligation Assay Antibodies
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The following antibodies were used for western blot detection: Anti-MBP mouse monoclonal antibody (from NEB, #E8032S, 1:10,000 dilution), Anti-GST rabbit monoclonal antibody (from Cell Signalling Technology, #2625S, 1:1,000 dilution) anti-Rac1/2/3 rabbit antibody (Cell Signalling Technology, #2465). Antibodies used for Proximity Ligation Assay: mouse monoclonal anti-FLAG M2 (Sigma-Aldrich, #F3165, 1:800 dilution), rabbit monoclonal anti-HA tag (Cell Signalling Technology, #3724, 1:800 dilution). DAPI was used to stain for nucleic acid (Thermo Scientific, #62248, 1:1,000 dilution).
7
Western Blot Analysis of Purified huPON2
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Purified huPON2 variants were analyzed by SDS-PAGE and transferred (1h at 100V) onto PVDF membranes in 0.375 M Tris-glycine buffer (pH8.3). The membranes were blocked by incubation with skim milk 5% (w/v) in 1x Trisbuffered saline (0.2 M Tris pH 7.5, 1.5 M NaCl) containing 0.05% Tween (v/v) and incubated for 1 h with an anti-MBP mouse monoclonal antibody (NEB). After washing, the membranes were incubated for 1 h with anti-mouse IgG-peroxidase conjugate (NEB), and developed with a chemiluminescent substrate.
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