GDF11-like changes were introduced into a pRK5 plasmid containing wild-type full-length human GDF8 using site-directed mutagenesis. GDF8/GDF11 chimeric protein was transiently produced using HEK293T co-transfected with human furin in pcDNA4 using polyethylenimine MAX (Polysciences, Inc.). Transfection proceeded for 4 h in culture medium followed by exchange into serum-free media. Conditioned media were collected 72–96 h later and concentrated 10 times. The concentrated media were applied to a Superdex S200 column (Amersham Biosciences). Fractions containing the latent complex were pooled and concentrated. Initial protein concentration and subsequent normalization were done by quantification (ImageJ) of SDS-PAGE colloidal Coomassie-stained gels.
Superdex s200 column
Manufactured by Cytiva
Sourced in United Kingdom
The Superdex S200 column is a size exclusion chromatography column designed for biomolecule separation. It is capable of separating proteins, peptides, and other biomolecules based on their molecular size and shape.
Automatically generated - may contain errors
Lab products found in correlation
Polyethylenimine max, by Polysciences (1 mentions)
Smart system, by Cytiva (1 mentions)
Hek293f suspension grown cells, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Anti flag m2 affinity resin, by Merck Group (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
4 protocols using superdex s200 column
1
Generation of Recombinant GDF8/GDF11 Chimeric Protein
2
Purification and Detection of Rab Proteins
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The assay was performed as previously.32 (link) Pancreatic tissue cytosolic fractions were loaded onto a Superdex S200 column using the SMART system (Amersham Biosciences, Amersham, UK). The column was equilibrated in a buffer containing (mmol/L) 50 Tris/HCl (pH 7.5), 100 NaCl, 8 MgCl2, 2 EDTA, and 1 dithiothreitol, supplemented with 10 μmol/L GDP, at a flow rate of 50 μL/min. The samples were injected, and the material eluting between 1.2 mL and 3.3 mL was collected in 42 50-μL fractions. Ten-μL aliquots of the fractions were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (on 4%–20% gel), transferred to nitrocellulose membranes, and Rab9 and RabGDIα/β proteins identified by IB. All the IBs (Figure 3 for the full, uncropped IBs) were developed together. Fractions 1–5 and 39–42 had no proteins recognized by the antibodies; therefore, these fractions are not shown in the figures.
3
Purification of SbmA-Fab Complex
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For the generation of the SbmA-Fab complex for structural studies, monoclonal antibody S11-1 was digested and purified using the Pierce Fab Preparation Kit. The purified Fab S11-1 was dialyzed in 20 mM tris (pH 7.5) including 25 mM NaCl. The SbmA and Fab S11-1 were mixed in a 1:2 molar ratio, and excess Fab was separated from the SbmA-Fab complex by size-exclusion chromatography using a Superdex S200 column (Cytiva) in 20 mM tris-Cl (pH 8.0), 150 mM NaCl, and 0.03% DDM.
4
Affinity Purification of Protein Complexes
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MIER1 constructs were cloned into pcDNA3.0 expression vectors containing an amino-terminal 10xHis-3xFLAG tag followed by a TEV protease cleavage site. Full length HDAC1(aa:1–482) and BAHD1(aa:525–780) were cloned without affinity tags into the same vectors. Constructs were co-transfected in HEK293F suspension grown cells (Invitrogen) using polyethylenimine (PEI; Sigma) as a transfection reagent and harvested after 48 h. Cells were lysed in buffer containing 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate, 5% v/v glycerol, 0.4% v/v Triton X-100 and Complete EDTA-free protease inhibitor (Roche). The lysate was clarified by centrifugation and applied to Anti-FLAG M2 affinity resin (Sigma Aldrich) for 30 min and washed three times with 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate and 5% v/v glycerol and three times more with 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate and 0.5 mM TCEP before being cleaved by TEV protease overnight. The protein complexes were purified further by gel filtration using a Superdex S200 column (Cytiva) with buffer containing 25 mM Tris/HCl pH 7.5, 50 mM potassium acetate and 0.5 mM TCEP.
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