EGFP-3x Myc cDNA mRNA was in vitro transcribed from its cDNA cloned into pSP64 poly(A) using mMESSAGE mMACHINE (ThermoFisher, Waltham, MA). The mRNA was translated in the rabbit reticulocyte lysate system (Promega, Madison, WI) with or without 1 μM (final) MBP or MBP-TRAL proteins. Synthesized EGFP-3x Myc protein and MBP or MBP-TRAL proteins from the reaction were examined by immunoblot with anti-Myc (9E10; Covance, Princeton, NJ) or Anti-MBP antibody (Sigma-Aldrich, St. Louis, MO).
Anti mbp antibody
Manufactured by Merck Group
Sourced in United States
The Anti-MBP antibody is a laboratory reagent used to detect and quantify the presence of myelin basic protein (MBP) in biological samples. MBP is a key component of the myelin sheath that insulates nerve fibers in the central nervous system. The Anti-MBP antibody can be utilized in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, to analyze MBP levels in research applications.
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Lab products found in correlation
Mmessage mmachine, by Thermo Fisher Scientific (1 mentions)
Rabbit reticulocyte lysate system, by Promega (1 mentions)
Anti myc 9e10, by Fortrea (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Glutathione sepharose 4b beads, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Anti o4, by Merck Group (1 mentions)
Anti mbp, by Merck Group (1 mentions)
Anti olig2, by Merck Group (1 mentions)
Sb203580, by Thermo Fisher Scientific (1 mentions)
Poly 1 c, by Thermo Fisher Scientific (1 mentions)
Percoll solution, by GE Healthcare (1 mentions)
Anti gfap antibody, by Merck Group (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Anti gst, by Merck Group (1 mentions)
Glutathione agarose beads, by Merck Group (1 mentions)
8 protocols using anti mbp antibody
1
EGFP-3x Myc Protein Translation
2
Myelination Regulation via PLGA-LBP Scaffold
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Myelination of Schwann cells is critical for the success of peripheral nerve regeneration. To evaluate the influence of PLGA-LBP nanofibrous scaffold on modulating myelination of Schwann cells, the expression of MBP of Schwann cell was determined with immunostaining. The process of immunostaining was the same as what described in section of Neuronal differentiation of PC12 cells. Anti-MBP antibody produced in rabbit (diluted at 1:200, Sigma) was used as the primary antibody in this study.
3
Protein-Protein Interaction Assay
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The coding sequences of GC1 and gc1-a were cloned into the C terminal of GST tag fusion pGEX-6p-1 vector. The cDNA of SbpPLAII-1 was cloned into the C-terminal of MBP tag fusion pMal-c2X vector. Each of purified recombinant bait protein GST, GST-GC1 and GST‐gc1-a, independently along with MBP-SbpPLAII-1 were both added to the binding buffer (50 mM Tris-HCl, 100 mM NaCl, 0.6% Triton X‐100, 0.2% glycerol, and 1 mM PMSF). The mixture was incubated overnight at 4 °C. Glutathione Sepharose 4B beads (GE, 17-0756-01) were added. Samples were incubated for 2 h at 4 °C, and subsequently washed with the binding buffer at least five times. The pulled‐down proteins by Glutathione Sepharose 4B beads were detected by immunoblotting with anti-MBP antibody (Sigma, M6295).
4
Immunoblotting Detection of MBP-AtSBT1.9
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The MBP-AtSBT1.9 fusion protein was expressed in E.coli using the conditions described above. Total bacterial protein was prepared and fractionated on 12% (v/v) SDS-PAGE and transferred to nitrocellulose membranes. Immunodetection was performed using an anti-MBP antibody produced in mice (Sigma-Aldrich, USA) against MBP-AtSBT1.9 at a 1:5,000 dilution (v/v) as the primary antibody, and using alkaline phosphatase-conjugated anti-mouse immunoglobulins (1:10,000 dilution, v/v) as the secondary antibody (Sigma-Aldrich). Signals were visualized by employing NBT (nitroblue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt) in the phosphatase reaction.
5
Oligodendrocyte Differentiation Markers
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Anti-O4, anti-MBP, anti-Olig2, and anti-S-100 antibodies were purchased from EMD Millipore (Billerica, Massachusetts, USA). CD68 (ED1) was purchased from ADb Serotech (Raleigh, NC). Anti MBP antibody was purchased from Millipore (Billerica, MA) and anti-CC1 antibody was purchased from Abcam (Cambridge, MA). SYBR Green master mix solution was purchased from Thermo scientific (Swedesboro, NJ). Triiodothyronine (T3), lysolecithin (LPC) and chloroquine were purchased from Sigma-Aldrich (St. Louis, MO). SB203580 and poly-IC was purchased from Invitrogen (San Diego, CA). Recombinant rat IL-33 was purchased from ProSpec-TanytechnoGen (East Brunswick, NJ). Bovine-FGF and PDGF were purchased from Pepro Tech (Rocky Hill, NJ). Anti-IL33 antibody was purchased from Enzo Life Sciences, Inc (Farmingdale, New York). Alexa-488 or 555 conjugated anti-rabbit or anti-mouse second antibodies were purchased from Life technologies (Grand Island, NY). Percoll solution was purchased from GE Healthcare Bio-Sciences (Pittsburgh, PA).
6
Evaluating White Matter Injury via Myelin and Astrogliosis
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White matter injury was evaluated by myelin basic protein (MBP) staining for myelination and glial fibrillary acidic protein (GFAP) staining for astrogliosis. After permealization and blocking of non-specific binding, sections were first incubated at 4°C overnight with the primary rat monoclonal anti-MBP antibody (1:100; Millipore, Billerica, MA, USA) or rabbit polyclonal anti-GFAP antibody (1:800; Millipore, Billerica, MA, USA), rinsed, and then incubated with biotinylated goat anti-rat (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-rabbit (1:300; Pierce Biotechnology, Rockford, IL, USA) IgG. Positively-stained cells were visualized using avidin-biotin-peroxidase complex amplification (Pierce Biotechnology, Rockford, IL, USA) with diaminobenzidine tetrahydrochloride detection. The MBP expression was assessed in 3 regions within the white matter in each hemisphere of each section at 100× magnification per visual field (0.579 mm2), and graded using a 4-point scoring system: 0, loss of processes and complete loss of the capsule; 1, loss of processes with thinning or breaks in the capsule; 2, complete loss of processes with intact capsule; 3, partial loss of processes; and 4, no MBP loss [8 (link),9 (link)]. The scores of each region were summed up to obtain a total score (range, 0 to 12) for each hemisphere.
7
Isolation and Knockdown of Rat OPCs
8
Protein-Protein Interaction Assays
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The plasmids for expressing the fusion proteins were generated using different primers (listed in Supplemental Table 6), and full-length CBF1/2/3 were cloned into the pMAL-c2 or pMAL-c5x vector and fulllength PIF3, PIF4 and PIF5 were cloned into the pET28a or pGEX4-1 vectors. The constructs were transformed into Escherichia coli BL21 or BL21 codon plus, and expression of the PIF3 and CBF1/2/3 fusion proteins was induced at 37 C for 3 h using 0.5 mM IPTG (isopropyl b-D-1-thiogalactopyranoside). Pull-down assays were performed using maltose binding protein agarose or glutathione-agarose beads (Sigma-Aldrich), and the pulled-down proteins were analyzed with anti-His, anti-GST or anti-MBP antibodies (Sigma-Aldrich).
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