Gradient-purified CASV virions were resuspended in the glycoprotein denaturing and reaction buffers (NEB) as per the manufacturer’s instructions, with or without the addition of 500 units PNGase F. Each preparation was incubated at 37°C for 1 h, prior to the addition of LDS sample buffer (Life Technologies) and separation on a 4–12% Bis-Tris SDS-PAGE gel (Life Technologies). The gel was stained using Sypro Ruby stain (Life Technologies) according to the manufacturer’s protocol and the protein bands were visualized on the Typhoon phosphoimager (GE Healthcare).
Glycoprotein denaturing and reaction buffers
Manufactured by New England Biolabs
Glycoprotein denaturing and reaction buffers are laboratory reagents designed to facilitate the study of glycoproteins. These buffers are used to denature and solubilize glycoproteins, allowing for further analysis and characterization.
Automatically generated - may contain errors
Lab products found in correlation
Lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Typhoon phosphoimager, by GE Healthcare (1 mentions)
Sypro ruby stain, by Thermo Fisher Scientific (1 mentions)
Bis tris sds page gel, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti mouse igg h l, by LI COR (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
2 protocols using glycoprotein denaturing and reaction buffers
1
Glycoprotein Denaturation and N-Glycan Removal
2
Castlerea Virus Protein Detection
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CsV-infected C6/36 lysate was harvested in NP-40 lysis buffer and clarified by centrifugation at 10 000g for 10 minutes at 4°C. Lysate was resuspended in glycoprotein denaturing and reaction buffers (NEB) as per the manufacturer’s instructions, with or without the addition of 500 U PNGase F. Each preparation was incubated at 37°C for 1 hour, prior to the addition of LDS sample buffer (Life Technologies), separation on a 4% to 12% Bis-Tris SDS-PAGE gel (Life Technologies), and transfer to a nitrocellulose membrane. Castlerea virus proteins were detected by incubation with CsV-immune mouse serum followed by IRDye 800CW Goat anti-Mouse IgG (H+L) (LI-COR) and visualised on the Odyssey imaging system (LI-COR, Nebraska, USA).
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