Journal:

Article Title: Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain *

doi: 10.1074/jbc.M900812200

Figure Lengend Snippet: Purification of Orai1 protein from transiently expressed HEK293 cells. N-terminally FLAG-tagged Orai1 was solubilized using DDM, then purified with immunoaffinity chromatography followed by SEC. A, SDS-PAGE of aliquots at each step in anti-FLAG affinity chromatography visualized by silver staining, and B, Western blotting using anti-FLAG antibody. Orai1 proteins are demonstrated as glycosylated (indicated as Orai1*, 44 kDa) and unglycosylated (Orai1, 36 kDa) forms. C, SEC profile using Superdex 200 column. The Orai1-rich eluates from the immunoaffinity column were concentrated using the Microcon YM-100 filter unit and analyzed by Superdex 200 SEC. Orai1 was mainly eluted in a peak at 1.04 ml elution (indicated by an arrow). Another peak at 1.96 ml contains FLAG-peptide. D, SDS-PAGE analysis of the SEC fractions, visualized by silver staining. Intensity of the bands of Orai1 in the gel corresponds well to the absorbance in SEC. The peak fraction at 1.04 ml (arrow) was used for electron microscopic analysis.

Article Snippet: The eluate containing Orai1 protein was concentrated 15 times with a Microcon centrifuge filter unit YM-100 (Millipore, Billerica, MA) and further purified by Superdex 200 (3.2/30) size exclusion chromatography (SEC) in a Smartsystem (GE Healthcare) using buffer A.

Techniques: Purification, Chromatography, SDS Page, Affinity Chromatography, Silver Staining, Western Blot