Aug 14, 2020
Universal Immunoblot analysis for investigating Protein-AG (SpAG)-binding to avian and mammalian immunoglobulins.
- 1University of the West Indies St. Augustine
Protocol Citation: Angel A Justiz-Vaillant 2020. Universal Immunoblot analysis for investigating Protein-AG (SpAG)-binding to avian and mammalian immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjseknbe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
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Created: August 14, 2020
Last Modified: August 14, 2020
Protocol Integer ID: 40486
Abstract
A protein that combines the binding capacity of SpA and SpG is not comercially available. It can be easy created in the laboratory by combining these two immunoglobulin-binding proteins to horseradish peroxidase by the periodate method [1]. However, a mixture of SpA and SpG could have the same effect as universal reagent in immunodetection.
1. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175
Aliquots of egg yolks, animal sera or 2 µg/µl of purified immunoglobulins from birds, laboratory, wild, farm animals and pets are applied to the gels of SDS-PAGE as described elsewhere.
Gels are transferred to nitrocellulose membranes (Immobilon-Nc, pore size 0.45 µm, Sigma-Aldrich Co, St Louis, Missouri) during 71 minutes at 40 mAmps using a semi-dry electroblotter, HEP-1 Model, Owl Scientific Inc.
The running buffer contains 25 mM Tris, 192 mM glycine pH 8.3 and 20% methanol.
The nitrocellulose membranes are blocked overnight in 10% non-fat skim milk in PBS with 0.05% Tween-20 pH 7.4 and then washed 4x, 10 minutes with PBS-Tween 20.
A mixture of SpA and SpG at a concentration of 5 µg/ml is added to the membranes. A recombinant Protein-AG (SpAG) could be used instead.
After that there is an incubation period of 12 hours at 4°C. It may be an overnight incubation period.
The nitrocellulose membranes were washed as above.
A secondary antibody (rabbit anti-chicken IgY horseradish peroxidase, Sigma Aldrich) is added at a 1:15 000 dilution.
It is incubated for one hour at room temperature and washed as above.
Tetramethyl-benzidine (TMB) solution is added to the nitrocellulose membranes, which are then incubated in the dark for seven minutes. Then, the membranes are shaken gently and rinsed thoroughly in de-ionized water to stop the blotting process and are left to dry.
Alternatively, Ig samples are transferred to nitrocellulose membranes and directly probed using SpAG-HRP (diluted 1:5000) and then adding TMB (this system was mainly used for detecting avian Igs).