Anti-PEG IgG1 (CH2074 except excess biotin experiment, which used CH2076, Silver Lake Research) and anti-PEG IgM (AGP4, IBMS) were used as test Ab and anti-Biotin IgG1 (Z021, ThermoFisher) and anti-Vancomycin IgM (2F10, Santa Cruz) were used as control Ab. All native Ab were used as is provided by the manufacturer. To prepare deglycosylated Ab, N-glycans on anti-PEG IgG and IgM were removed with rapid non-reducing PNGase F enzyme (New England Biolabs) according to the manufacturer’s protocol. The deglycosylation reaction was confirmed with two NuPAGE 4–12% Bis-Tris gels (Novex) in MOPS buffer. The first gel was transferred to a nitrocellulose membrane (Novex) with a Semi-Dry Blotter (Novex), blocked with carbo-free buffer (Vector Labs), labeled overnight at 4°C in 2 µg/mL biotinylated concavalin A (Vector), and probed with anti-biotin peroxidase (Vector) before imaging with Clarity Western Blot ECL Substrate (BioRad) in a FluorChemE unit (Cell Biosciences). The second gel was silver stained with Pierce Silver Stain Kit (Thermo Scientific) and imaged with the same unit. The remaining deglycosylated IgG and IgM was buffer-exchanged into PBS using 50 MWCO spin-x columns (Corning) and quantified based on A280.
Biotinylated concavalin a
Manufactured by Vector Laboratories
Biotinylated concavalin A is a lectin that binds to glycoproteins containing alpha-mannose and alpha-glucose residues. It is commonly used in various biotechnological applications as an affinity reagent for the isolation and detection of glycosylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti biotin igg1, by Thermo Fisher Scientific (1 mentions)
Semi dry blotter, by Thermo Fisher Scientific (1 mentions)
Carbofree buffer, by Vector Laboratories (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Clarity ecl western blot substrate, by Bio-Rad (1 mentions)
2 protocols using biotinylated concavalin a
1
Deglycosylation of Anti-PEG Antibodies
2
Biofilm Polysaccharide Staining in Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Biotinylated-Concavalin A (B-1005, Vector Laboratories) was used to stain biofilm polysaccharides. Fixed brains were washed three times 10 min in PBS and incubated 1 h at room temperature with PBS containing 0.1% Tween 20 and 1% BSA. Brains were then incubated overnight at 4 °C with ConA at 1/200 in blocking solution (PBS containing 0.1% Tween 20 and 1% BSA). Brains were then washed with PBS containing 0.1% Tween before 3 h incubation with Streptavidin-A488 at 1/300 in blocking solution (PBS containing 0.1% Tween 20 and 1% BSA). Brains were washed with PBS-Tween 0.1%, mounted in Mowiol mounting medium and visualised with a laser scanning confocal microscope (Zeiss LSM 880 with Zen software (2012 S4)).
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