Description
Detailed procedure
After cell lysis DYKDDDDK-tagged proteins are magnetically labeled with µMACS Anti-DYKDDDDK MicroBeads. The sample is loaded onto a MACS Column placed in the magnetic field of a µMACS or thermoMACS Separator in which the magnetically labeled DYKDDDDK-tagged and associated proteins are retained during the washing steps. After elution with denaturing elution buffer, the precipitated proteins can be analyzed by SDS-PAGE and Western blot. Alternatively, native proteins can be eluted by pH-shift using triethylamine pH 11.8 or by removing the column from the magnetic field and applying a suitable native elution buffer.
Applications
The µMACS and MultiMACS DYKDDDDK Isolation Kits facilitate direct immunoprecipitation of DYKDDDDK fusion proteins from cell lysates at an analytical scale. They can also be used in co-immunoprecipitation experiments for the isolation of associated proteins or protein complexes in protein-protein interaction studies.
Downstream applications
The (co-)immunoprecipitated proteins can be analyzed by SDS-polyacrylamide gel electrophoresis, Western blot analysis, or mass spectrometry. Enzymatic on-column assays can also be performed using the thermoMACS Separator.
Overview
The µMACS and MultiMACS DYKDDDDK Isolation Kits facilitate the isolation of DYKDDDDK-tagged (also known as FLAG®-tagged) proteins from different sources. The super-paramagnetic µMACS MicroBeads included in the kits are conjugated to an anti-DYKDDDDK monoclonal antibody, enabling fast and effective magnetic labeling of DYKDDDDK-tagged fusion proteins. The protocol allows for protein isolation within just 1.5 hours. No centrifugation or buffer removal steps are required.
