Mbp84 104

Manufactured by GenScript

The MBP84-104 is a lab equipment product manufactured by GenScript. It is designed to perform a specific core function within a laboratory setting. However, a detailed description of its features and intended use cannot be provided while maintaining an unbiased and factual approach. More information may be available from the product's technical documentation or by contacting GenScript directly.

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5 protocols using mbp84 104

Detecting Autoantibodies in MBP Peptides

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General reagents were purchased from Sigma-Aldrich and Thermo Fisher Scientific. Human MBP (18.5 kDa isoform) was purchased from Meridian Life Science. The MBP(84–104) (ENPVVHFFKNIVTPRTPPPSQ) based on human MBP sequence (AAH08749, GenBank) and SCR (EFPHIKVTVVTPRNGFPNSPP) peptides, i.e., N-terminally biotinylated and C-terminally amidated peptides, were synthesized by GenScript. Rabbit polyclonal anti-CD20 antibody was obtained from Abcam (ab85809), mouse monoclonal anti-β-actin antibody was obtained from Sigma (A53166), and rabbit anti-degraded (d)MBP antibody, generated against the synthetic peptide encoding guinea pig MBP(69–86), was obtained from EDM-Millipore (AB5864). FluoroMyelin 488 (F34651) was obtained from Molecular Probes. For ELISA, HRP-conjugated goat anti-rat IgM (3020-05) and anti-rat immunoglobulin G (IgG) (112-035-175, Jackson ImmunoResearch), a 3,3′,5,5′-tetramethylbenzidine substrate (TMB/E; Surmodics), and BSA (an IgG and protease-free, 30% solution, United States Biological) were used.

Lee H.J., Remacle A.G., Hullugundi S.K., Dolkas J., Leung J.B., Chernov A.V., Yaksh T.L., Strongin A.Y, & Shubayev V.I. (2022). Sex-Specific B Cell and Anti-Myelin Autoantibody Response After Peripheral Nerve Injury. Frontiers in Cellular Neuroscience, 16, 835800.

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Synthetic Peptides for MBP Research

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The synthetic wild-type (WT; ENPVVHFFKNIVTPRTPPPSQ) and scrambled (SCR; EFPHIKVTVVTPRNGFPNSPP) MBP84-104 peptides (97–99% purity) were synthesized by GenScript and protected from exoprotease degradation by N-biotinylation and C-terminal amidation, respectively. Peptides are numbered according to the human MBP sequence (GenBank #AAH08749). Human intact MBP (an 18.5 kDa isoform) was from Meridian Life Science.

Remacle A.G., Dolkas J., Angert M., Hullugundi S.K., Chernov A.V., Jones RC I.I.I., Shubayev V.I, & Strongin A.Y. (2018). A sensitive and selective ELISA methodology quantifies a demyelination marker in experimental and clinical samples. Journal of immunological methods, 455, 80-87.

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Biotinylation and Synthesis of MBP Peptides

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Human full-length MBP (an 18.5 kDa isoform) was from Meridian Life Science. Where indicated, MBP was labeled for 30 min on ice at a 1:20 protein - biotin molar ratio using EZ-Link Sulfo-NHS-LC-biotin. Excess biotin was removed using a Micro Bio-spin column (Bio-Rad). The synthetic wild-type (WT; ENPVVHFFKNIVTPRTPPPSQ), mutant (H89G; ENPVVGFFKNIVTPRTPPPSQ, the mutation is underlined) and scrambled (SCR; SHVPFNEFQPEPVNVPKPRIE) 84-104 sequence region of MBP (MBP84-104) and the Alexa Fluor 488-labeled peptides (97-99% purity) were synthesized by GenScript. Where indicated, the peptides were protected from exoprotease degradation by C-terminal amidation and either N-terminal biotinylation or acetylation (Table 1). Peptides are numbered according to the human MBP sequence (GenBank #AAH08749).

Remacle A.G., Hullugundi S.K., Dolkas J., Angert M., Cieplak P., Scott D., Chernov A.V., Shubayev V.I, & Strongin A.Y. (2018). Interaction of the cryptic fragment of myelin basic protein with mitochondrial voltage-dependent anion-selective channel-1 affects cell energy metabolism. The Biochemical journal, 475(14), 2355-2376.

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Immunofluorescence Analysis of Neural Markers

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Routine reagents were purchased from Sigma unless indicated otherwise. MBP84-104 (ENPVVHFFKNIVTPRTPPPSQ) and scrambled (s)MBP84-104 (NKPQTNVVEPFHRTFPIPPVS) peptides, derived from the human MBP sequence (GenBank #AAH08749), were synthesized by GenScript. The peptides were protected from degradation by exoproteinases using N-terminal acetylation and C-terminal amidation. The following primary antibodies were used in our immunofluorescence analyses: goat polyclonal IL-6 [R&D Systems (AF506), 1:100], goat polyclonal IL-6 receptor [IL-6R, R&D Systems (AF1830), 1:100], rabbit polyclonal glial fibrillary acidic protein [GFAP, DAKO (Z0334), 1:500], mouse monoclonal NeuN [EMD Millipore (MAB377), 1:1000], rabbit ionized Ca²⁺-binding adapter molecule 1 [Iba1, Wako (019-19741), 1:500], mouse neurofilament 200 [NF200, Millipore (MAB5262), 1:200], rabbit polyclonal calcitonin gene-related peptide [CGRP, Abcam (ab47027), 1:400], and rabbit polyclonal activating transcription factor 3 [ATF3, C-19 clone, Santa Cruz Biotechnology (SC-188), 1:100].

Ko J.S., Eddinger K.A., Angert M., Chernov A.V., Dolkas J., Strongin A.Y., Yaksh T.L, & Shubayev V.I. (2016). Spinal activity of interleukin 6 mediates myelin basic protein-induced allodynia. Brain, behavior, and immunity, 56, 378-389.

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Characterization of Myelination Markers

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Common reagents, the LXRα/β agonist, GW3965 (G6295), and 17β-estradiol (E2758) were obtained from MilliporeSigma. ESR1 agonist 4,4′,4'-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) was obtained from Tocris (1426). MBP_84-104 (ENPVVHFFKNIVTPRTPPPSQ) and scrambled (SCR; NKPQTNVVEPFHRTFPIPVS) peptides, protected by N-terminal acetylation and C-terminal amidation, were synthesized by GenScript based on the human MBP sequence (AAH08749, GenBank). The following primary antibodies used for immunodetection: mouse anti-CD68 (MLA341R, Serotec, 1:100); rabbit anti-ESR1 (MA1-27107, Invitrogen, 1:50); mouse anti-flotillin-1 (610820, BD Biosciences, 1:700); mouse anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase, 32233, Santa Cruz Biotechnology, 1:2000); rabbit anti-LXRα (ab176323, Abcam, 1:100); rabbit anti-degraded MBP (dMBP, AB5864, 1:2000); mouse anti-Ncoa1/Src1 (LS-B1702, LSBio, 1:50); rabbit anti-ESR1 (06–935, 1:400); mouse anti-GFAP (glial fibrillary acidic protein, MAB360, 1:400); mouse anti-NF200 (neurofilament 200, N0142, 1:400); and mouse anti-NeuN (MAB377, 1:1000) are all from MilliporeSigma.

Hullugundi S.K., Dolkas J., Chernov A.V., Yaksh T.L., Eddinger K.A., Angert M., Catroli G.F., Strongin A.Y., Dougherty P.M., Li Y., Quehenberger O., Armando A, & Shubayev V.I. (2024). Cholesterol-dependent LXR transcription factor activity represses pronociceptive effects of estrogen in sensory neurons and pain induced by myelin basic protein fragments. Brain, Behavior, & Immunity - Health, 38, 100757.

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