The ActRIIA-Fc and ActRIIB-Fc proteins described in this report were expressed in stably transfected Chinese hamster ovary cells and were generated at Eli Lilly (Indianapolis, IN, USA). Isolation of the chimeric proteins from concentrated cell culture supernatants was performed using a two-step purification method. In the first step, crude conditioned cell culture media containing the specific variant was captured onto Mab select sepharose (GE Healthcare, Buckinghamshire, UK) under high salt conditions (1 M sodium chloride) and eluted using a step-gradient of 10 mM sodium citrate, pH 3.0. Pooled protein was concentrated using an Amicon Ultra-15 concentrator (Millipore, Boston, MA, USA) and further purified using a Superdex G200 preparative gel-filtration step (GE Healthcare, Buckinghamshire, UK). These steps generally resulted in protein purity of > 95%, as assessed by SimplyBlue staining (Invitrogen, Carlsbad, CA, USA) SDS-PAGE and analytical gel filtration on a TSKG3000SWXL column (Tosoh Bioscience, Tokyo, Japan). GDF8 antibody was generated by Eli Lilly and Company and its properties were described previously [30 (link),31 (link)]. Activin A antibody was purchased from R&D System (Cat #MAB3381, Minneapolis, MN, USA).
Simplyblue staining
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
SimplyBlue is a Coomassie-based protein stain designed for visualizing proteins in polyacrylamide gels. It provides a simple, fast, and sensitive staining method without the use of methanol or acetic acid. SimplyBlue stains proteins with a blue color, allowing for easy detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Glutathione sepharose bead, by Thermo Fisher Scientific (2 mentions)
Amicon ultra 15 concentrator, by Merck Group (1 mentions)
Tsk g3000swxl column, by Tosoh (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Xcell surelock mini cell electrophoresis system, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
4 protocols using simplyblue staining
1
Purification of ActRIIA-Fc and ActRIIB-Fc Proteins
2
Investigating SMN-U1-70K Protein Interactions
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Recombinant GST-U1-70K proteins (2.5 μg) were immobilized on the glutathione-Sepharose beads (Invitrogen) in 500 μL RSB-200 buffer containing 0.02% Triton X-100. After washing unbound proteins, 5 μg of recombinant or in vitro-translated [35S]- methionine-labeled SMN proteins were incubated for 1 hr at 4 °C and then washed with the binding buffer four times. The bound proteins were eluted by boiling in 1X sample buffer and resolved by SDS-PAGE and visualized by SimplyBlue staining (Invitrogen), silver staining, or autoradiography. The band intensities were analyzed using Bio-Rad Quantity One software. Binding experiments were performed side-by-side with the same protein preparations and run on the same gel. Uncropped images of gels, autoradiographs or western blots are provided in Supplementary Data Set 1 .
3
Investigating SMN-U1-70K Protein Interactions
4
Protein Purity Analysis by SDS-PAGE
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Protein purity was analysed by SDS-PAGE on 4–12% Bis-Tris NuPAGE gels (Invitrogen Ltd, Paisley, UK) using XCell SureLock™ Mini-Cell Electrophoresis System (Invitrogen Ltd, Paisley, UK) and NuPAGE MES SDS running buffer (Invitrogen Ltd, Paisley, UK). All samples were heated prior to loading at 70 °C for 10 min in NuPAGE LDS sample buffer (Invitrogen Ltd, Paisley, UK). Gels were typically run at 200 V for 45 min. After electrophoretic separation, proteins were visualised by SimplyBlue staining (Invitrogen Ltd, Paisley, UK). The Perfect Protein molecular weight standard (Merck, Darmstadt, Germany) was used as marker.
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