Aug 14, 2020
Immunoblot analyses for investigating SpLA binding to purified mammalian and avian immunoglobulins.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Immunoblot analyses for investigating SpLA binding to purified mammalian and avian immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjq7kmzn
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
We use this protocol and it's working
Created: August 14, 2020
Last Modified: August 14, 2020
Protocol Integer ID: 40447
Aliquots of 2 µg/µl 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 75 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.
Recombinant protein LA (Sigma-Aldrich Co, St Louis, Missouri) at a concentration of 5 µg/ml is added to the membranes.
After that there is an incubation period at 4°C overnight.
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 a few 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 SpLA-HRP (diluted 1:5000) and TMB (this system was mainly used for detecting avian Igs).