To generate whole cell lysates, cells were washed with PBS and lysed in RIPA buffer. Nuclear fraction proteins were isolated using a NE-PER Nuclear Protein Extraction Kit (Thermo Scientific) according to the manufacturer’s instructions. For the detection of secreted Wnt7a, 48 h-conditioned media were collected and filtered with a Millex-GP Syringe Driven Filter Unit (Millipore, cat# SLGP033NS) to exclude cells and debris and were then concentrated to 100 μl by centrifugation at 6000 RPM for 30 min with Amicon Ultra-15 Centrifugal Filters with Ultracel-10K membranes (Millipore, cat# UFC901008). The primary antibodies are listed in Supplementary Table 1 , and the signals were detected with the LICOR® Odyssey Infrared Imaging System after incubation with appropriate IRDye® secondary antibodies (LICOR Biosciences).
Amicon ultra 15 centrifugal filters with ultracel 10k membranes
Manufactured by Merck Group
The Amicon Ultra-15 Centrifugal Filters with Ultracel-10K membranes are a laboratory filtration device. The device uses centrifugal force to separate and concentrate molecules, cells, or other particulates from liquid samples based on their molecular weight or size.
Automatically generated - may contain errors
2 protocols using amicon ultra 15 centrifugal filters with ultracel 10k membranes
1
Whole Cell Lysate and Secreted Protein Extraction
2
Whole Cell Lysate and Secreted Protein Extraction
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To generate whole cell lysates, cells were washed with PBS and lysed in RIPA buffer. Nuclear fraction proteins were isolated using a NE-PER Nuclear Protein Extraction Kit (Thermo Scientific) according to the manufacturer’s instructions. For the detection of secreted Wnt7a, 48 h-conditioned media were collected and filtered with a Millex-GP Syringe Driven Filter Unit (Millipore, cat# SLGP033NS) to exclude cells and debris and were then concentrated to 100 μl by centrifugation at 6000 RPM for 30 min with Amicon Ultra-15 Centrifugal Filters with Ultracel-10K membranes (Millipore, cat# UFC901008). The primary antibodies are listed in Supplementary Table 1 , and the signals were detected with the LICOR® Odyssey Infrared Imaging System after incubation with appropriate IRDye® secondary antibodies (LICOR Biosciences).
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