IP with different antibody formats

Conventional Abs used for IP are typically monoclonal or polyclonal immunoglobulin G (IgG) isolated from rabbit or mouse. They are ~150 kDa in size and composed of two heavy (2x ~50 kDa) and two light (2x ~25 kDa) chains. The most common and straightforward option for immobilizing Abs on beads for IP is to bind the Ab via its Fc-fragment to protein A or G beads. When using protein A or G beads, either the cell lysate is first incubated with the Ab and then the beads are added, or the Ab is pre-loaded onto the beads, which are then added to the cell lysate. Analysis of the IP on SDS-PAGE and WB show additionally to the POI also the heavy and light chain of the Ab.

Advantages:

• During immobilization of the Ab on protein A or G, the paratope (binding region) of the Ab is usually neither modified, unfolded, nor denatured and the Ab remains intact.

Limitations:

• The heavy and light chains of the Ab are visible on SDS-PAGE and WB; this complicates identification or can mask the POI and its interaction partners.
• Abs are sensitive to harsh buffer ingredients such as detergents, reducing agents, and salts, which may lead to the release of the POI during washing steps.
• Protein A or G and the large Ab molecule offer more surface for unwanted interactions with the cellular proteome or other contaminants, which increases the background of the IP.
• Immobilization of the Ab on protein A or G beads involves two incubation steps; these are time-consuming and require optimization and greater hands-on time.

Fab-fragments are derived from monoclonal Abs from mouse or rabbit. They are composed of the light chain (~25 kDa) as well as the V_H and C_H1 domain of the heavy chain (~25 kDa), making ~50 kDa in total. ChromoTek’s  Fab-Trap™  is an IP reagent that consists of a Fab-fragment, covalently bound to beads. Despite the short equilibration, no additional pre-incubation steps are required in the preparation procedure of the Fab-Trap™. Also, the Fab-fragment is co-eluted upon boiling in SDS sample buffer. However, due to the reduced size of the Fab-fragment, only one additional band at ~25 kDa is visible on subsequent SDS-PAGE and WB. The band is composed of the light chain as well as the heavy chain fragment.

Advantages:

• Due to the lack of the Fc-fragment, the Fab-Trap™ generates fewer contaminating Ab bands on SDS-PAGE and WB and no bands above ~25 kDa. Elution of the Fab-fragment can be avoided by applying milder elution conditions such as low pH or peptide elution.
• Compared to full-size Abs, the Fab-Trap™ tolerates increased concentrations of detergents and reducing agents, which enables more stringent washing.
• Due to the smaller size of the Fab-fragment and the lack of protein A or G, the Fab-Trap™ provides less surface for unwanted interactions. This leads to lower background compared to IP with full-size Abs.
• The Fab-Trap™ is ready to use and requires less hands-on time and no incubation with protein A or G beads.
• The Fab-Trap™ is produced recombinantly, which results in virtually no lot-to-lot variations and higher reproducibility.

Limitations:

• The additional band on SDS-PAGE and WB at ~25 kDa can mask the POI and interaction partners.

Nanobodies are single-domain antibody fragments derived from alpaca heavy chain only antibodies. They consist of the V_H-domain of the heavy chain (also called V_HH) and are around 15 kDa in size. ChromoTek’s  Nano-Trap  is an IP reagent that consists of a Nanobody, covalently bound to beads. Nano-Traps are ready to use and require only a short equilibration step, with no need for additional pre-incubation, e.g., with protein A or G beads. During elution, the Nanobody of the Nano-Trap remains bound to the beads and does not appear in SDS-PAGE or WB analyses.

Advantages:

• On SDS-PAGE and WB only the POI band is visible, with no contamination of the Nano-Trap.
• Nano-Traps offer the highest purity for IP as Nanobodies are the smallest molecules and bind with high specificity.
• Nano-Traps can withstand very high concentrations of detergents, reducing agents, and salts, enabling the highest stringency during washing and IP even under denaturing conditions. Therefore, Nano-Traps offer the lowest background among IP reagents.
• Nano-Traps are ready to use, do not need to be pre-incubated, and therefore require less hands-on time.
• Due to the recombinant production of the Nanobodies, a limitless supply of Nano-Traps is possible, with no lot-to-lot variation, leading to more reproducible results.

Limitations:

• Due to their preference for native and structurally intact epitopes, the binding of Nano-Traps to fixed samples may be challenging.