Amicon ultra 50 kda mwco spin filter columns

Sortase A was expressed in E.coli using plasmid pET28a-SrtAdelta59 (Addgene #51138)^{48 (link)}, and purified using Ni-NTA coated agarose beads (ThermoFisher). A photocleavable blocking peptide was synthesized (Biopeptide Inc.) with three N-terminal glycine residues followed by a photocleavable linker (Santa Cruz Biotechnology) and anti-FLAG or Cetuximab blocking peptide. Anti-FLAG or Cetuximab antibodies photo-conjugated with PpL fused to the Sortase recognition site, were diluted to 1 µM in TBS with 10 mM CaCl₂ along with approximately 50 µM purified Sortase and 200 µM synthetic peptide. This was left to react overnight at room temperature, after which antibodies were purified using Amicon Ultra 50 kDa MWCO spin filter columns (Millipore-Sigma, UCFC505008).

In initial screens, PpL mutants and anti-CD3 (clone UTH1, BD bioscience 555329) antibodies were diluted into PBS pH7.6 such that the final concentrations were approximately 50 µM and 2 µM, respectively, and loaded into thin walled 200 µL polypropylene microtubes (PCR tubes). This mixture was then irradiated for 1 h under 365 nm light at an intensity of 6.4 mW/cm² from an LED source (M365LP1, Thor Labs) 14 cm away. Products were reduced using DTT solution (ThermoFisher) and separated on 4–12% BisTris PAGE gels (ThermoFisher) to observe photoconjugation. Full gel images are shown in Supplementary Fig. S2. Photocleavage was accomplished using the same irradiation setup. Photoconjugations to anti-FLAG antibody (anti-DYDDDDK clone 1557CT661.18.1, Lifespan Biosience LS‑C392574) or Cetuximab (Selleck Chemicals A2000) were done identically, with 100 µM PpL constructs that had been freshly purified. Photoconjugates were then purified from excess PpL using Amicon Ultra 50 kDa MWCO spin filter columns (Millipore-Sigma, UCFC505008). The unaltered antibody control conditions of our ELISA experiments, described below, validated Anti-FLAG binding to the FLAG peptide and Cetuximab binding to EGFR protein.