Cell lysates or conditioned media that were concentrated using Amicon Ultra 0.5 10K Centrifugal Filters (EMD Millipore, Billerica, MA, USA) were prepared using standard protocols. The following antibodies were used: AXL (4566), pAXL (5724), ALK (3333), pALK (3341), AKT (4691), pAKT (9271), ERK (4695), pERK (4377), VIM (5741), CDH1 (3195), tubulin (2128) and actin (4967) from Cell Signaling Technology (Danvers, MA, USA); GAS6 (sc-376087), HSP90 (sc-59577) and phosphotyrosine (sc-81529) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Flag (F3165) from Sigma-Aldrich (Saint Louis, MO, USA); and AXL antibody for immunocytochemistry from R&D Systems (AF154).
Amicon ultra 0.5 10k centrifugal filters
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra 0.5 10K Centrifugal Filters are a type of lab equipment used for sample preparation and concentration. They feature a 10,000 molecular weight cut-off (MWCO) membrane that allows the filtration and concentration of macromolecules such as proteins, peptides, and nucleic acids. The device can be used with a standard laboratory centrifuge to separate the desired molecules from smaller components in the sample.
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3 protocols using amicon ultra 0.5 10k centrifugal filters
1
Protein Expression Analysis in Cell Lysates
2
Protein Production Analysis via SDS-PAGE
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Supernatant was obtained from cultures grown for 72 h in YNB, YPD, or YP2D4 media and was concentrated tenfold in 30 mM Tris (pH 8.0) and 50 mM NaCl using Amicon Ultra-0.5 10 K centrifugal filters (Merck Millipore Ltd, Ireland). Protein production was analyzed via polyacrylamide gel electrophoresis (SDS-PAGE); 4–12% Tris–Glycine gels and an XCell SureLock™ Mini-Cell electrophoresis system (Novex, Life Technologies, Saint-Aubin, France) were used. Prism (MW1; 19–130 kDa) and wide-range (MW2; 14–212 kDa) protein molecular weight markers were used as standards (VWR Chemicals, Fontenay-sous-Bois, France). The gels were stained with 0.2% Coomassie Brilliant Blue R dye (Thermo Fisher Scientific, Villebon-sur-Yvette, France).
3
Protein Expression and Phosphorylation Analysis
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