Anti human immunoglobulin antibodies

To detect binding of purified anti-PsA peptide antibodies to TLR2, HEK cell lysates were prepared with the use of a commercially available kit (Nuclear Extract Kit, Active Motif, Carlsbad, CA United States). Cell lysates were immunoprecipitated with rabbit antibody directed against human TLR2 (Abcam) cross-linked to Sepharose. Eluted proteins were resolved by means of sodium dodecyl sulfate–polyacrylamide-gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, New Jersey, United States). Blots were probed with primary antibodies followed by peroxidase-linked anti-human immunoglobulin antibodies (Sigma). In another set of experiments HEK cell lysates were were resolved by SDS-PAGE and transferred onto nitrocellulose paper. Blots were probed with a commercially available monoclonal antibody directed against TLR2 (Abcam) followed by peroxidase-labeled anti-mouse immunoglobulin antibodies.

The antibody binding to the Arf GAP with the SH3 Domain, Ankyrin Repeat and PH Domain (ASAP1) protein was tested using the recombinant ASAP1, (OriGene Inc., Rockville, MD, USA) using an immunoblot assay. The blots were probed with primary antibodies (human anti-peptide antibodies or mouse control monoclonal antibody) followed by either peroxidase-conjugated anti-human immunoglobulin antibodies or mouse IgG antibodies (Sigma) (10 μg per millilitre). A monoclonal antibody against ASAP1 was purchased from OriGene and used as a positive control. A monoclonal antibody directed against beta-actin was used as an equal loading control (Abcam).
The blots were developed using chemiluminescence according to the manufacturer’s instructions (Thermo Scientific).