The traditional lectin FLISA is described elsewhere[5 ]. The modified assay involves incubation of 1–5 μl of human serum (diluted into 45–49 μl PBS) with 20 μl of Pierce™ Protein A/G Plus (Thermo Fisher, Waltham, MA) for 1 hour prior to filtration in a 100KD Amicon Ultran 0.5 mL spin filter (EMD Millipore, Billerica, MA). Flow through was applied directly to plates and the lectin FLISA performed as before[5 ]. For the anti-alpha 1 anti-trypsin (A1AT) Lectin-FLISA, a polyclonal anti-A1AT (Sigma-Aldrich, St. Louis, MO) was used.
Pierce protein a g plus
Manufactured by Thermo Fisher Scientific
The Pierce™ Protein A/G Plus is a purification resin designed for the efficient capture and purification of antibodies from various sample types. It combines the binding properties of Protein A and Protein G to provide a versatile solution for immunoglobulin purification.
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2 protocols using pierce protein a g plus
1
Modified Lectin-FLISA for Serum Analytes
2
Co-Immunoprecipitation of Protein Complexes
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Co-IP were performed as our previous description.14 (link) Precleared cell lysates were incubated with the indicated antibodies or control IgG under constant rotation at 4°C overnight. Antibodies against CD36 (sc-7309) and Src (sc-5266) were from Santa Cruz (Dallas, TX), and α7nAChR (ab216485) were from Abcam (Cambridge, MA). Then the protein extracts were incubated with the equilibrated Pierce Protein A/G Plus (ThermoFisher,Waltham, MA) at 4°C for 2 hours with gentle mixing to bind the specific antibody captured proteins. Immunoprecipitates captured with protein A/G-agarose were washed 3 times with the lysis buffer. Beads were finally collected and the bounded fusion proteins were eluted from the beads for further western blot analysis.
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