Total cell lysates were prepared as described in NP-40 buffer (1% NP-40, 150mM NaCl, 2mM EDTA, 50mM Tris-HCl, pH8.0). For the FLAG IP, proteins were incubated with anti-FLAG affinity gel (Sigma) for 1hour at 4°C with rotating. Beads were washed 4 times with wash buffer, and then resuspended in protein loading buffer. For the PAK4 IP, lysates were pre-cleared with Protein A Agarose (Invitrogen) for 1 hour at 4°C and then incubated with 10 μg of anti-PAK4 antibody (Cell Signaling), overnight at 4°C. Antigen-Antibody bound Protein A Agarose pellets were washed 4 times with wash buffer, and then resuspended in protein loading buffer. The released proteins were fractionated by Bis-Tris 4%–20% SDS-PAGE (Invitrogen), and then transferred to nitrocellulose membranes. Immunoblotting was performed as described above using antibodies RhoA, PAK4, Flag, or β-actin antibody as described above.
Bis tris 4 20 sds page
Manufactured by Thermo Fisher Scientific
Bis-Tris 4%–20% SDS-PAGE is a pre-cast polyacrylamide gel designed for the separation and analysis of proteins. It features a Bis-Tris buffer system and a gradient of 4% to 20% acrylamide concentration. This product is intended for use in standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques.
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2 protocols using bis tris 4 20 sds page
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Immunoprecipitation and Western Blot Analysis
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Immunoprecipitation and Western Blot Analysis
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Total cell lysates were prepared as described in NP-40 buffer (1% NP-40, 150mM NaCl, 2mM EDTA, 50mM Tris-HCl, pH8.0). For the FLAG IP, proteins were incubated with anti-FLAG affinity gel (Sigma) for 1hour at 4°C with rotating. Beads were washed 4 times with wash buffer, and then resuspended in protein loading buffer. For the PAK4 IP, lysates were pre-cleared with Protein A Agarose (Invitrogen) for 1 hour at 4°C and then incubated with 10 μg of anti-PAK4 antibody (Cell Signaling), overnight at 4°C. Antigen-Antibody bound Protein A Agarose pellets were washed 4 times with wash buffer, and then resuspended in protein loading buffer. The released proteins were fractionated by Bis-Tris 4%–20% SDS-PAGE (Invitrogen), and then transferred to nitrocellulose membranes. Immunoblotting was performed as described above using antibodies RhoA, PAK4, Flag, or β-actin antibody as described above.
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