Co-IP of BAX and BAK using anti-BAX IgGs from different vendors by anti-rabbit IgG/ anti-mouse IgG VHH Agarose (mrIGa). Tested IgGs include such of rabbit and mouse origin with IgG1 and IgG2b subtypes. 5 µg of respective IgG was spiked into HEK293T cell lysate derived from 0.5x10^7 cells. 1% of input (IN) and 25% of bound (B) fraction was loaded onto an SDS-PAGE gel. For Western Blot analysis BAX was detected using a polyclonal rabbit IgG (PTG: 50599-2-Ig) (1:2000) labeled using a conformation-specific HRP-conjugated secondary. The presence of BAK co-precipitated with BAX was tested using a polyclonal rabbit IgG (PTG 29552-1-AP) (1:2000). While all IgGs allowed for efficient IP of BAX, BAK was absent in the case of the primary antibody from vendor D, possibly due to the BAX:BAK interaction interface being masked by this particular IgG.

Co-IP of MCM complex via pulldown of MCM6 using 5 µg of anti-MCM6 antibodies and anti-rabbit IgG / anti-mouse IgG VHH agarose (mrIGa). All subunits of the 600 kDa heterohexameric complex are successfully precipitated using both a mouse IgG1 (67989-1-Ig) and a rabbit (13347-2-AP) MCM6 antibody, as shown by western blot anaylsis using subunit specific antibodies. Apparent molecular weights are provided. For Input (IN) and flow through (FT) fractions 1% was loaded. For bound (B) fraction and bound fraction of isotype control antibody (BISO), 20% was loaded. Product codes of PTG-antibodies are provided in parentheses. BISO is an isotype control used as negative control (ms1: 66260-1-Ig; rb: 30000-0-AP).

IP of PCNA using 5 µg of anti-PCNA antibody (IgG1, 60097-1-Ig) by anti-rabbit IgG/ anti-mouse IgG VHH agarose (mrIGa). For Western blot analysis the PCNA polyclonal antibody (10205-2-AP, 1:2000) was labeled using a conformation-specific HRP-conjugated secondary to avoid staining of heavy and light chain of the IP-antibody. In contrast to many competitor protein A and G beads, clean pulldown of PCNA by Proteintech anti-rabbit IgG/ anti-mouse IgG VHH agarose facilitates unambiguous identification of the target protein. Other beads show leaching of protein A or protein G fragments into the final IP fractions, which can lead to binding of the detection antibody and unspecific signals, as reported previously (Grant et al., 2019, Biiol Proced Online, doi: 10.1186/s12575-019-0095-z). For input (IN) and bound (B) fractions 1% and 30% were loaded, respectively.

IP of Alpha-2-Macroglobulin (A2M) from human serum using anti-rabbit IgG / anti-mouse IgG VHH agarose (mrIGa) and comparison to protein A and G beads. IP was performed using 5 μg of rabbit anti-A2M IgG (13545-1-AP) in 1:10 diluted human serum. 0.5% and 6.25% of input (IN) and bound (B) fractions were analyzed by SDS-PAGE, respectively. 5 μg of rabbit isotype control antibody (BISO,  98136-1-RR) was used as negative control. The Coomassie-stained gel shows precipitation of A2M by anti-rabbit IgG / anti-mouse IgG VHH agarose and reveals its absence for protein A and G beads. This is likely due to human IgGs competing with the spiked IP-antibody for the latter beads. Results were verified using Western blot analysis, which detected A2M at approximately 180 kDa using an anti-A2M primary IgG (13545-1-AP, 1:5000).

IP of GAPDH from common human cell lines using anti-rabbit IgG/ anti-mouse IgG VHH Agarose (mrIGa). IP was performed using 5 μg of rabbit anti-GAPDH IgG (10494-1-AP) with 1x107 cells used per IP reaction. 1% and 25% of input (IN) and bound (B) fractions were analyzed by SDS-PAGE, respectively. Bound fractions without antibody spiked (BBG) are shown as background control. The Coomassie-stained gel shows precipitation of GAPDH by anti-rabbit IgG / anti-mouse IgG VHH agarose, what was verified using Western blot analysis using the anti-GAPDH mouse monoclonal antibody (60004-1-Ig).