Chabc

Manufactured by Seikagaku

Sourced in Japan

ChABC is a laboratory enzyme product. It functions to cleave chondroitin sulfate proteoglycans.

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Lab products found in correlation

F4 80, by Bio-Rad (1 mentions) Cs 56, by Nichirei Biosciences (1 mentions) Histofine kit, by Nichirei Biosciences (1 mentions) Cs 56, by Seikagaku (1 mentions) Chabc, by MP Biomedicals (1 mentions) Pnase, by Merck Group (1 mentions) Peptide n glycosidase f pngase f, by Roche (1 mentions) Pa 03 column, by YMC (1 mentions) Pd 10 column, by GE Healthcare (1 mentions)

6 protocols using chabc

Quantifying Chondroitin Sulfate Digestion

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Immunohistochemistry was used to stain for the C4S-stub antigen that remains following ChABC-mediated digestion. Staining for C4S (mouse monoclonal; 1:500; MPBio #636511) was completed using sections adjacent to the TH-stained sections whilst using the same protocol as previously described for TH [29 (link)].
To confirm whether the extent of ChABC-mediated digestion of CS-GAGs along the nigrostriatal pathway in vivo was maximal, the effect of subsequent ChABC exposure ex vivo was assessed. Briefly, 7 µm SNc brain sections from saline- and ChABC-treated mice from both the full and partial lesion studies (n = 3 for each group) were incubated with ChABC (10 U/ml; Seikagaku) or tris-buffered saline for 3 h at 37 °C. C4S-stub immunoreactivity was then stained for, as described above.

Fletcher E.J., Moon L.D, & Duty S. (2019). Chondroitinase ABC reduces dopaminergic nigral cell death and striatal terminal loss in a 6-hydroxydopamine partial lesion mouse model of Parkinson’s disease. BMC Neuroscience, 20, 61.

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Post-Operative Nerve Regeneration Interventions

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For the BPI group, rats were intraperitoneally injected with 5% alcohol-saline solution 5 minutes after surgery, and 1, 2, 3, and 4 hours after surgery. A gelatin sponge was used to absorb 10 μL saline and to cover the C6 nerve root after replantation. For the MT group, rats were intraperitoneally injected with 2.5 mg/kg MT (Sigma, St Louis. MO, USA), and 5% alcohol-saline solution 5 minutes after surgery and 1, 2, 3, and 4 hours after surgery (Yang et al., 2015). A gelatin sponge was used to absorb 10 μL saline and to cover the C6 nerve root after replantation. For the ChABC group, rats were intraperitoneally injected with 5% alcohol-saline solution 5 minutes after surgery and 1, 2, 3, and 4 hours after surgery. A gelatin sponge was used to absorb 10 μL ChABC 2.5 U/mL (Seikagaku Corporation, Tokyo, Japan) and saline and to cover the C6 nerve root after replantation (Yick et al., 2000). For the ChABC + MT group, rats were intraperitoneally injected with 2.5 mg/kg MT (Sigma), and 5% alcohol-saline solution 5 minutes after surgery and 1, 2, 3, and 4 hours after surgery (Yang et al., 2015). A gelatin sponge was used to absorb 10 μL ChABC 2.5 U/mL (Seikagaku Corporation) and saline and to cover the C6 nerve root after replantation (Yick et al., 2000).

Guo W.L., Qi Z.P., Yu L., Sun T.W., Qu W.R., Liu Q.Q., Zhu Z, & Li R. (2019). Melatonin combined with chondroitin sulfate ABC promotes nerve regeneration after root-avulsion brachial plexus injury. Neural Regeneration Research, 14(2), 328-338.

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Chondroitin Sulfate Glycosaminoglycan Staining Protocols

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Immunohistochemical staining was performed as previously reported [4 (link), 22 (link)]. The following first Abs were used: 3B3 (Seikagaku Corporation, Tokyo, Japan) for paraffin embedded sections, F4/80 (Serotec, Oxford, UK) and CS-56 (Seikagaku Corporation) for frozen sections [23 (link)]. 3B3 recognizes an epitope created following chondroitinase ABC (ChABC) degradation of chondroitin-6 sulfate. CS-56 recognizes an epitope on some intact chondroitin sulfate glycosaminoglycan chains. The number of F4/80⁺ cells per mm² of alveolar wall was determined using light microscopy, in at least 30 randomly chosen high-power fields [4 (link)]. Immunolabeling for CS-56 and 3B3 were used Histofine kit (Nichirei Bioscience, Tokyo, Japan) according to the manufacturer’s instructions. Tissue sections for 3B3 were pre-incubated in vitro with 0.2 U/mL ChABC (Seikagaku Corporation) in Tris–HCl, pH 8.0, for 1 h at 37 °C.

Kai Y., Tomoda K., Yoneyama H., Yoshikawa M, & Kimura H. (2015). RNA interference targeting carbohydrate sulfotransferase 3 diminishes macrophage accumulation, inhibits MMP-9 expression and promotes lung recovery in murine pulmonary emphysema. Respiratory Research, 16, 146.

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Stereotaxic Chondroitinase Delivery in Rat Perirhinal Cortex

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Protease-free ChABC (Seikagaku, Japan) or penicillinase (P-nase; Sigma-Aldrich, UK) as a non-relevant control enzyme was dissolved in 0.1% BSA to 50 U/ml of concentration and filtered through a 0.2 micron filter. Six injections in the PRh (3 per hemisphere, 0.5 μl with a speed of 0.2 μl/min) were performed stereotaxically under isoflurane anesthesia with a 10 μl Hamilton syringe and a 33 gauge needle at the following sites (in mm from bregma and the surface of skull): 1. anterior–posterior (AP): − 1.8; lateral (L): ± 4.6; ventral (V): − 4.4, 2. AP: − 2.8; L: ± 4.8; V: − 4.3 and 3. AP: − 3.8; L: ± 4.8; V: − 3.8. The needle remained in place at the injection site for 3 min before being slowly withdrawn over 2 min.

Yang S., Cacquevel M., Saksida L.M., Bussey T.J., Schneider B.L., Aebischer P., Melani R., Pizzorusso T., Fawcett J.W, & Spillantini M.G. (2015). Perineuronal net digestion with chondroitinase restores memory in mice with tau pathology. Experimental Neurology, 265, 48-58.

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Enzymatic Deglycosylation of Soluble Brain Proteins

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Soluble fraction proteins (30 μg) from mouse brains were precipitated with ethanol and suspended with 20 to 50 μl of chondroitinase buffer (10 mM Tris-HCl (pH7.4), 30 mM sodium acetate, 50 mM EDTA and protease inhibitors), followed by incubation with a final concentration of 250 μU/ml of chondroitinase ABC (chABC) (Seikagaku Corporation) for 3 h at 37°C. The reaction mixtures were denatured with 0.5% sodium dodecyl sulfate (SDS) for 5 min at 100°C. To reduce the SDS concentration, the solution was diluted with 4 volumes of phosphate-buffered saline (PBS) containing 25 mM EDTA, 1.25% NP-40 and 1.25% 2-mercaptoethanol. Peptide N-glycosidase F (PNGase F, Roche Applied Science) was added at the final concentration of 20 mU/μl, followed by incubation for 16 h at 37°C.

Yabuno K., Morise J., Kizuka Y., Hashii N., Kawasaki N., Takahashi S., Miyata S., Izumikawa T., Kitagawa H., Takematsu H, & Oka S. (2015). A Sulfated Glycosaminoglycan Linkage Region Is a Novel Type of Human Natural Killer-1 (HNK-1) Epitope Expressed on Aggrecan in Perineuronal Nets. PLoS ONE, 10(12), e0144560.

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Quantitative Analysis of Aggrecan Disaccharides

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The purified recombinant aggrecan-Fc was subjected to gel filtration on a PD-10 column (GE Healthcare). The flow-through fractions were collected and digested with 5 mIU of chABC (Seikagaku Corporation) in 60 mM sodium acetate, 50 mM Tris-HCl (pH 8.0) for 12 h at 37°C. The digests were derivatized with a fluorophore, 2-aminobenzamide, and then analyzed by anion-exchange high-performance liquid chromatography (SLC-10A, Shimadzu) on a PA-03 column (YMC). Identification and quantification of the resulting disaccharides were achieved by comparison with chondroitin sulfate-derived authentic unsaturated disaccharides (Seikagaku Corporation), as described previously [38 (link)].

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