Observations on different resin strategies for affinity purification mass spectrometry of a tagged protein.
Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins. We observed significant variability for isolation of tagged bait proteins between Protein A/G Agarose, Protein G Dynabeads, and AminoLink resins. While previous research identified the importance of tag sequence and their location, crosslinking procedures, reagents, dilution, and detergent concentrations, the effect of the resin itself has not been considered. Our data suggest the type of solid support is important and, under the conditions of our experiments, AminoLink resin provided a more robust solid-support platform for AP-MS.
The University of Houston, Department of Biology and Biochemistry, 4800 Calhoun Road, SERC 4022, Houston, TX 77204, USA.
Sujina
S
Mali
The University of Houston, Department of Biology and Biochemistry, 4800 Calhoun Road, SERC 4022, Houston, TX 77204, USA.
Wilna J
WJ
Moree
The University of Houston, Department of Biology and Biochemistry, 4800 Calhoun Road, SERC 4022, Houston, TX 77204, USA.
Morgan
M
Mitchell
The University of Houston, Department of Biology and Biochemistry, 4800 Calhoun Road, SERC 4022, Houston, TX 77204, USA.
William
W
Widger
The University of Houston, Department of Biology and Biochemistry, 4800 Calhoun Road, SERC 4022, Houston, TX 77204, USA. Electronic address: sbark@uh.edu.
Steven J
SJ
Bark
D000906:Antibodies / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000737:chemistry*
D000906:Antibodies; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A
Affinity purification; Agarose; AminoLink; Crosslinking; Dynabeads; Mass spectrometry; Solid phase resins