1×107 wild type and CRISPR-Cas9 edited monoclonal cells were resuspended in 200 μl of 2× Laemmli sample buffer [100 mM Tris-Cl (pH 6.8), 4% (w/v) sodium dodecyl sulfate (SDS), 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol and 200 mM dithiothreitol] and heated at 95°C for 10 minutes. 2–20 μl of cell lysate was separated by SDS-PAGE, transferred to nitrocellulose membrane and block with 5% non-fat milk in 1× PBS containing 0.05% Tween-20. Membranes were then probed with α-Tubulin antibody (1:20,000–1:500,000, Sigma) and the appropriate primary antibody (α-PMP70 (1:500, Abcam), PEX5 (1:1,000, Novus), or α-PEX19 (1:500, ThermoFisher Scientific) for 6 hrs at RT or overnight at 4°C followed by secondary antibody-conjugated horseradish peroxidase (HRP) for 1–2 hrs at RT. Proteins were detected with Amersham ECL™ Start Western Detection Reagent (GE Healthcare) and imaged using a AI600 Gel Imaging System (GE Healthcare).
α pmp70
Manufactured by Abcam
α-PMP70 is a protein that functions as a peroxisomal membrane protein. It is involved in the transport of various molecules across the peroxisomal membrane.
Automatically generated - may contain errors
2 protocols using α pmp70
1
Quantitative Western Blot Analysis of Peroxisomal Proteins
2
Antibody Sourcing for Cell Biology
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Rabbit polyclonal α-PMP70 was purchased from Abcam. Rabbit polyclonal α-NBR1 was a gift from I. Dikic (Goethe Univerity, Frankfurt, Germany). Human polyclonal α-P0 was a gift from A. Palazzo (University of Toronto, Toronto, Canada). Mouse monoclonal α-TOMM20 was purchased from Santa Cruz Biotechnology, Inc. Mouse monoclonal α-Mfn2 was purchased from Cedarlane. Rabbit monoclonal α-calnexin was a gift from D. Williams (University of Toronto, Toronto, Canada). Mouse monoclonal α-HA was purchased from Covance. Rabbit polyclonal α-Atg12 was purchased from Cell Signaling Technology. Rabbit polyclonal α-PEX14 was purchased from Millipore. Rabbit polyclonal α-PEX2 was generated from peptide by GenScript. Rabbit polyclonal α-PEX2 was purchased from Abcam. Rabbit polyclonal α-PEX5 was generated by GenScript. Rabbit polyclonal α-catalase was purchased from EMD Millipore. Rabbit monoclonal α-4E-BP was a gift from J. Brumell (University of Toronto, Toronto, Canada). Mouse monoclonal α-FLAG was purchased from Sigma-Aldrich.
HRP-conjugated goat polyclonal α-mouse secondary and HRP-conjugated goat polyclonal α-rabbit secondary was purchased from Cedarlane. HRP-conjugated mouse monoclonal α-GAPDH was purchased from Abcam. Fluorescent goat polyclonal α-rabbit Alexa Fluor 568 secondary antibody and goat polyclonal α-mouse Alexa Fluor 488 secondary antibody were purchased from Invitrogen.
HRP-conjugated goat polyclonal α-mouse secondary and HRP-conjugated goat polyclonal α-rabbit secondary was purchased from Cedarlane. HRP-conjugated mouse monoclonal α-GAPDH was purchased from Abcam. Fluorescent goat polyclonal α-rabbit Alexa Fluor 568 secondary antibody and goat polyclonal α-mouse Alexa Fluor 488 secondary antibody were purchased from Invitrogen.
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