Commercial IgA1 or purified IgA1 from IgAN or non-IgAN patient serum or artificial IgA1-IgG immune complexs was resolved in 12% SDS-PAGE gel and transferred to PVDF membrane (Minipore, USA). After 1-hour’s blocking with 2.5% bovine serum albumin, HRP-conjugated mouse anti-human IgA1 monoclonal antibody (Fc fragment specific, 1:1000, Abcam, USA) or HRP-conjugated donkey anti-goat IgG (1:1000, Santa Cruze, USA) was employed to recognize the total IgA1 at 4 °C overnight. The membrane was then washed with TBST to remove unbound antibody followed by signal development with Immobilon Western Chemiluminescent HRP Substrate (Minipore, USA). For detection of galactose-deficient IgA1 by western blot, the blot was firstly treated with 15 mU/ml neuraminidase for 6 hours at 37 °C after membrane transfer. After washing with TBST, biotinylated HAA (1:200, Sigma, USA) was used to bind the GalNAc-exposed IgA1 overnight at 4 °C and HRP-conjugated avidin (1:5000, Sigma, USA) was introduced to recognize biotin for 1 hours at 37 °C. Signal development was the same as above. Gray intensity of western blot signal band was calculated with Image J 1.47V software (NIH, USA).
Biotinylated haa
Manufactured by Merck Group
Sourced in United States, France
Biotinylated HAA is a laboratory reagent used in various biochemical and molecular biology applications. It functions as a labeled molecule that can be detected or captured using streptavidin-based techniques. The core purpose of Biotinylated HAA is to serve as a versatile tool for researchers working in fields such as protein analysis, immunoassays, and affinity purification.
Automatically generated - may contain errors
2 protocols using biotinylated haa
1
Quantification of Galactose-Deficient IgA1 by Western Blot
2
Quantifying Terminal Galactosyl-N-acetylamine in Serum IgA1
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The glycan content of serum IgA1 was measured with enzyme-linked immunosorbent assay using HAA, a lectin that binds to terminal galactosyl-N-acetylamine residues. 19 Briefly, Microtitration plates (Maxisorp, Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 10 μg/ml anti-human IgA (Bethyl Laboratories, Montgomery, TX, USA). After 8 h of blocking, the serum samples were adjusted to 10 μg/ml of IgA and incubated in the plate overnight at 4 °C. Neuraminidase (from V. Cholerae, Roche, Boulogne-Billancourt, France) was added or not in the plate at 10 mU/ml in a sodium acetate buffer, pH = 5, to remove sialic acid for 3 h at 37 °C. Biotinylated HAA (Sigma-Aldrich, Saint-Quentin Fallavier, France; 1/100) was added to wells for 3 h at 37 °C. After 30 min incubation with streptavidin-alkaline phosphatase, the alkaline phosphatase substrate was added, and plates were read at 405 nm. The lectin reactivity was expressed as the ratio of sample optical density (OD) to positive control OD. The positive control was a purified IgA1 in which galactose and neuraminic acid had been enzymatically removed earlier.
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