Binding of SAA1 to the LRP1 ectodomain was confirmed by size exclusion chromatography. Purified SAA1 was incubated with retinol at a molar ratio of 1:1.5 at 4°C in the dark for 20 min to allow formation of the SAA1–retinol complex, followed by passage over a Superdex 75 10/300 GL size exclusion chromatography column (GE Healthcare) to remove the excess retinol. Next, the SAA1-retinol complex was mixed with the purified full-length ectodomain of LRP1 at a molar ratio of 10:1 in buffer containing 10 mM MES pH 5.5 and 100 mM NaCl. The mixture was incubated on ice for 1 hour and then loaded onto a Superose 6 increase 10/300 GL column (GE Healthcare). As controls, LRP1 ectodomain and SAA1–retinol were individually analyzed in the same way.
Superdex 75 10 300 gl size exclusion chromatography column
Manufactured by GE Healthcare
The Superdex 75 10/300 GL is a size exclusion chromatography column used for the separation and purification of proteins, peptides, and other biomolecules based on their molecular size. It has a separation range of 3,000 to 70,000 daltons and a column volume of 24 mL. The column is packed with a stable, cross-linked agarose-dextran matrix and is designed for use with low- to medium-pressure liquid chromatography systems.
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6 protocols using superdex 75 10 300 gl size exclusion chromatography column
1
Characterization of SAA1-Retinol Binding to LRP1
2
UbcH5B~Ubiquitin Conjugation Protocol
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UbcH5B~ubiquitin linkage was performed based on published methods21 (link). Briefly, Ube1, UbcH5B(S22R/C85K) and ubiquitin were mixed and buffer exchanged into 50 mM Tris pH 10, 150 mM NaCl using PD-10 desalting columns (GE Healthcare). 10 mM MgCl2, 5 mM ATP and 1 mM TCEP were added and the protein was incubated at 37°C for 16 h. The completeness of the reaction was monitored using SDS-PAGE and covalently linked UbcH5B~ubiquitin was purified from unreacted proteins and Ube1 using a Superdex 75 10/300 GL size exclusion chromatography column (GE Healthcare) equilibrated in protein buffer.
3
Purification and Characterization of His-Tagged Proteins
4
UbcH5B~Ubiquitin Conjugation Protocol
5
Stable Oxyester-linked UBCH5B-Ubiquitin Complex
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For experiments using oxyester-linked UBCH5B-ubiquitin complexes, an active site Cys-to-Ser mutation (C85S UBCH5B) was performed. The resulting bond was only one atom different from the wt thioester and significantly more stable. A successful conjugation reaction was typically accomplished by mixing 1 µM E1, 150–200 µM E2, 500–600 µM ubiquitin, cycling buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7), 10 mM creatine phosphate (Sigma-Aldrich), ~0.6 units mL−1 creatine kinase (Sigma-Aldrich), 5 mM MgCl2, and 5 mM ATP. Conjugation reactions were incubated at 37 °C for 16–20 h, and the reaction was monitored using SDS–PAGE. Oxyester linked UBCH5B-ubiquitin was purified from E1 and unreacted proteins using a Superdex® 75 10/300 GL size-exclusion chromatography column (GE Healthcare) equilibrated in 50 mM K2HPO4 and 50 mM KH2PO4 pH 7 [24 (link)]. The purified C85S UBCH5B–Ub conjugate was collected, flash-frozen in liquid nitrogen and stored at −80 °C.
6
Synthesis of Oxyester-linked UbcH5B-Ubiquitin Conjugates
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For experiments using oxyester-linked UbcH5B–ubiquitin complexes, an active site Cys-to-Ser mutation (UbcH5B C85S) was performed. The resulting bond was only one atom different from the wt thioester and significantly more stable. A successful conjugation reaction was typically accomplished by mixing 1 µM E1, 150–200 µM E2, 500–600 µM ubiquitin, cycling buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7), 10 mM creatine phosphate (Sigma-Aldrich), ~0.6 units mL−1 creatine kinase (Sigma-Aldrich), 5 mM MgCl2, and 5 mM Adenosine Triphosphate (ATP). Conjugation reactions were incubated at 37 °C for 16–20 h, and the completeness of the reaction was monitored using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Oxyester-linked UbcH5B–ubiquitin was purified from E1 and unreacted proteins using a Superdex® 75 10/300 GL size-exclusion chromatography column (GE Healthcare) equilibrated in 50 mM K2HPO4 and 50 mM KH2PO4 pH 7 buffer. The purified C85S UBCH5B–Ub conjugate was collected, flash-frozen in liquid nitrogen, and stored at −80 °C. The same protocol was performed in the synthesis of C85S-S22R UbcH5B-Ub conjugate.
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