To determine if rAAS27 was N- and/or O-glycosylated, 5 μg of affinity-purified rAAS27 was treated with deglycosylation enzyme mix according to manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Deglycosylation was verified resolving proteins on a 12% SDS-PAGE followed by western blotting analysis using an antibody directed to C-terminus hexa histidine-tag (Life Technologies, Carlsbad, CA, USA) at 1:5,000 dilution and positive signal detected using HRP chromogenic substrate (Thermo Scientific, Waltham, MA, USA).
Hrp chromogenic substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HRP chromogenic substrate is a laboratory reagent used to visualize the presence and localization of horseradish peroxidase (HRP) in various bioanalytical applications. It is a chromogenic substrate that undergoes a color change reaction when catalyzed by the HRP enzyme, enabling the detection and quantification of target analytes.
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Lab products found in correlation
5 protocols using hrp chromogenic substrate
1
Deglycosylation of Recombinant Protein rAAS27
2
Analyzing Glycosylation of rAAS19
3
Glycosylation Analysis of Eukaryotic Proteins
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Post translational glycosylation in eukaryotes is involved in promoting protein folding and improves protein stability thereby functional roles of proteins. Amino acid sequences of AAS 41 and 46 were scanned for potential N-linked and O-linked glycosylation sites using the software NetNGlyc 1.0 Server [47] and NetOGlyc 4.0 Server [48] , respectively. Scanning AAS 41 and 46 amino acid sequences revealed three N-linked and six O-linked glycoslyation sites and thus to confirm if functional, affinity purified rAAS 41 and 46 were treated with protein deglycosylation enzyme mix according to manufacturer's instructions (New England Biolabs, Ipswich, MA, USA). Deglycosylation was verified by western blotting analysis using an antibody against the C-terminus hexa histidine-tag (Thermo Fisher Scientific) and the positive signal detected using HRP chromogenic substrate (Thermo Fisher Scientific).
4
Western Blot Protein Detection
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Supernatants from transfected cells were subjected to 4–12% Bis-Tris sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein bands in the gel were transferred to polyvinylidene difluoride membranes (Invitrogen). The membranes were incubated with HRP-conjugated goat anti-mouse IgG (H+L) (Pierce), and immunoreactive bands were visualized using a HRP chromogenic substrate (Invitrogen).
5
Recombinant Rrd1 Protein Purification
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12 percent polyacrylamide gel was used to transfer the purified recombinant Rrd1 protein PVDF membrane for 90 minutes at 50 mV. With 5% skim milk, the protein-transfer membrane was stopped for three hours. After that, the membrane was cleaned and treated with a Penta-His Antibidy, BSA free (QIAGEN) 1:1000 (100 ng/ul) in PBS contain 1% BSA. The membrane washed then treated with Secondary Antibody (Promega Corporation) Goat Anti-Mouse IgG HRP Conjugate (1mg/ml) washed three times with PBS, pH 7.4 PBST (PBS,.05 percent Tween). Chromomeric Substrate (HRP Conjugate) (Invitrogen Novex HRP Chromogenic Substrate) was used to develop the membrane [22 (link), 24 (link)].
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