While traditional resin-based methods of immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) are well-characterized and routinely used, they are often time-consuming and laborious. Transmembrane proteins, even when highly expressed, can pose a problem. Researchers at the University of Bern have found our Capturem IP & Co-IP Kit, featuring a high-capacity Protein A membrane, to be an excellent alternative. This method is capable of efficiently recovering difficult cardiac voltage-gated channel proteins.

Using the Capturem IP & Co-IP Kit resulted in efficient immunoprecipitations of both channels, and efficient co-immunoprecipitation of the Ca_v1.2-α2δ1 subunit with Ca_v1.2-α1c. This highlights the quality of this technology for performing in-vivo immunoprecipitations using lysates from challenging samples such as mouse ventricular heart tissue.

—Sabrina Guichard, Hugues Abriel, and Jean-Sébastien Rougier of the Institute of Biochemistry and Molecular Medicine at the University of Bern

Efficient immunoprecipitation of cardiac voltage-gated channel proteins

Voltage-gated ion channels are transmembrane proteins that play critical roles in exciting and modulating muscle cells throughout the body, including cardiac muscles. The similarly structured sodium and calcium channels are key players that enable contraction of the heart. The channels consist of multiple subunits; a primary subunit forms the pore and auxiliary subunits regulate function.

IP of voltage-gated sodium (Na_v1.5) and calcium (Ca_v1.2-α1c) channels, and Co-IP of auxiliary calcium channel subunit Cav1.2-α2δ1, were performed using the Capturem IP & Co-IP Kit. Lysates were created from ventricles of adult mice, ~40 weeks old. IP was performed after incubation with or without the antibody against Na_v1.5 (Panel A), or Ca_v1.2 (Panel B), respectively. Immunoprecipitated fractions were then visualized on western blots using an antibody against Na_v1.5 (Panel A), Ca_v1.2-α1c (Panel B, upper section), or Ca_v1.2-α2δ1 (Panel B, lower section). Input lanes contain whole heart lysate (without IP procedure) showing the presence of the proteins of interest.

As observed in Panels A and B, use of the Capturem IP & Co-IP Kit resulted in efficient IP of both channels, and efficient Co-IP of the Ca_v1.2-α2δ1 subunit with Ca_v1.2-α1c. These data highlight the benefit of employing Capturem technology when performing IP of lysates from challenging samples such as mouse ventricular heart tissue.

The Capturem IP & Co-IP Kit improves upon traditional resin-based methods, offering efficient IP and Co-IP of cardiac voltage-gated ion channel proteins. Experiments are streamlined down to just five minutes of hands-on time while maintaining excellent results. The protocol's speed saves antibody-protein complexes from possible degradation and/or loss of activity that might result from lengthy resin-based procedures.

To learn more about how Capturem Protein A columns combine the specific antibody-binding properties of Protein A with novel Capturem technology, see our tech note, Fast, efficient, and specific immunoprecipitation and co-immunoprecipitation with Capturem Protein A technology.

Immunoprecipitation of Na_v1.5

• Two hearts (left and right ventricles) were harvested from mice around 40 weeks old.
• Protein extracts were made using a homemade lysis buffer.
• Protein aliquots of 3.75 mg were incubated with anti-Na_v1.5 antibody (Alomone labs, cat. #AGP-008) at 0.95 mg/ml (40 μl = 38 μg).
• IP was performed using the Capturem IP & Co-IP Kit according to the kit's instructions.

Western blot of Na_v1.5

• Na_v1.5 bands were visualized using homemade rabbit anti-Na_v1.5 at a 1:200 dilution.

Immunoprecipitiation of Ca_v1.2-α1c

• One heart (left and right ventricles) was harvested from a mouse around 40 weeks old.
• Protein extracts were made using homemade lysis buffer.
• Protein aliquots of 3.75 mg were incubated with anti-Ca_v1.2-α1c antibody (Alomone Labs, cat. #ACC003) at 0.80 mg/ml (20 μl = 16 μg).
• IP and Co-IP were performed using the Capturem IP & Co-IP Kit according to the kit's instructions.

Western blot of Ca_v1.2-α1c

• Ca_v1.2-α1c bands were visualized using mouse anti-Ca_v1.2-α1c (UC Davis/NIH NeuroMab clone N263/31) at a 1:1,000 dilution.
• Cav1.2-α2δ1 bands were visualized using mouse anti-Cav1.2-α2δ1 (Abcam, cat. #ab2864) at a 1:400 dilution.