Rabbit IgG (anti beta-tubulin; 80713-1-RR) was conjugated with NHS-Azide (lane 1) and then with DBCO-Oligo (lane 2). Post conjugation clean-up was done using CleanAb Kit for rabbit IgG (lanes 3-5). Excess oligo was successfully removed as shown in SYBR Gold DNA staining and IgG eluted with high recovery (~70%) at neutral pH (lane 4). Desalting was done with Zeba spin desalting columns (lane 5). Only very little IgG remaining on beads (lane 6).

Rabbit IgG (Isotype control, lane 1) was bound to CleanAb rabbit beads (flowthrough in lane 2), then functionalized on beads with NHS-Azide (flowthrough in lane 3) and then with DBCO-Oligo (flowthrough in lane 4). Excess oligo was successfully removed as shown in SYBR Gold DNA staining and the oligo-conjugated IgG was eluted with high recovery at neutral pH (lane 6). Desalting was done with Zeba spin desalting columns (lane 8). Only very little IgG remaining on beads (lane 9). Note: the CleanAb Elution Buffer can interfere with SDS gels, which can make the CleanAb elution appear washed out.

Rabbit IgG (anti beta-tubulin; 80713-1-RR) was conjugated with NHS-Azide (lane 1) and then with DBCO-Oligo (lane 2). (left) Post conjugation clean-up was done using CleanAb Kit for rabbit IgG (lanes 3-5). Excess oligo was successfully removed as shown by SYBR Gold DNA staining and IgG eluted with high recovery (~70%) at neutral pH (lane 4). Desalting was done with Zeba spin desalting columns (lane 5). Only very little IgG remaining on beads (lane 6) (right) Post conjugation clean-up was done using Protein A beads (lanes 3-5). Excess oligo was successfully removed as shown by SYBR Gold DNA staining but IgG elution with low pH failed (lane 4). Most IgG remains on beads (lane 6).

(left) Rabbit IgG (Isotype control, lane 1) was bound to CleanAb rabbit beads (flowthrough in lane 2), then functionalized on beads with NHS-Azide (flowthrough in lane 3) and then with DBCO-Oligo (flowthrough in lane 4). Excess oligo was successfully removed as shown in SYBR Gold DNA staining and the oligo-conjugated IgG was eluted with high recovery at neutral pH (lane 6). Desalting was done with Zeba spin desalting columns (lane 8). Only very little IgG remaining on beads (lane 9). Note: the CleanAb Elution Buffer can interfere with SDS gels, which can make the CleanAb elution appear washed out. (right) Rabbit IgG (Isotype control, lane 1) was bound to Protein A beads (flowthrough in lane 2), Excess oligo was successfully removed as shown in SYBR Gold DNA staining but the oligo-conjugated IgG failed to elute at low pH (lane 6). Most IgG remains on beads (lane 9).

Immunostaining of HeLa cells with rabbit anti-CD147 antibodies (11989-1-AP, 1:100, orange hot), chemically conjugated to a 15-nt DNA oligonucleotide and purified using CleanAB beads. Detection was performed by hybridization with an imaging DNA strand labeled with ATTO643. Nuclei were counterstained with DAPI (cyan). Direct comparison of CleanAb product and desalted product demonstrates functionality of both products. While signal intensity is lower for the desalted sample, successful staining can still be achieved.

Immunostaining of HeLa cells with rabbit anti-CD147 antibodies (11989-1-AP, 1:100, orange hot), chemically conjugated to a 15-nt DNA oligonucleotide and purified using CleanAB beads. Detection was performed by hybridization with an imaging DNA strand labeled with ATTO643. Nuclei were counterstained with DAPI (cyan). Direct comparison of the two possible CleanAb protocols (post conjugation clean-up and on bead conjugation & clean-up) shows that both approaches are viable and will yield functional antibody-oligo conjugate.