Preparation of 10% SDS polyacrylamide gels and protein electrophoresis were carried out according to Laemmli [25] (link). For Western blot analysis proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA), probed with primary antibodies and detected with HRP-conjugated secondary antibodies and the ECL-Plus Kit (GE Healthcare, Little Chalfont, UK). Primary antibodies were directed against cMyc (Roche), Cox2p (cytochrome c oxidase subunit 2; Invitrogen, Carlsbad, CA), Aco1p (aconitase; kind gift of R. Lill), Pgk1p (phosphoglycerate kinase; Invitrogen), Dpm1p (dolichol phosphate mannose synthase; Invitrogen), Cit1p (citrate synthase; kind gift of T. Fox), Ccp1p (cytochrome c oxidase; kind gift of W. Neupert), Tom40p (subunit of the outer membrane translocase; kind gift of J. Brix) and lipoic acid (LA; Calbiochem).
Dpm1p
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Dpm1p is a laboratory equipment that measures the disintegration of radioactive materials. It provides accurate and reliable data on the radioactive decay of samples.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus kit, by GE Healthcare (1 mentions)
Cox2p, by Thermo Fisher Scientific (1 mentions)
C myc, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Porin, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Irdye conjugated secondary antibody, by LI COR (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
4 protocols using dpm1p
1
SDS-PAGE and Western Blot Analyses
2
Organelle Membrane Purity Evaluation
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The isolation and purity of the organelle membranes was monitored by immunoblot analysis with polyclonal rabbit antibodies raised against Pcs60p (Blobel and Erdmann, 1996 (link)), Cta1p and Fox1p (Schafer et al., 2004 (link)), Tom40p and Tim23p (from K. Pfanner, University of Freiburg), Por1p and Kar2p (from R. Rachubinski, University of Alberta), Fbp1p (Entian et al., 1988 (link)) and mouse antibodies raised against Dpm1p (Invitrogen, Karlsruhe, Germany). Primary antibodies were detected with an IRDye 800CW goat anti-rabbit or anti-mouse IgG secondary antibody (Li-Cor Bioscience, Bad Homburg, Germany).
3
Protein Visualization and Analysis
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Proteins were extracted from cells grown to an OD550 of 1.0, separated by 4–12% SDS-PAGE (Invitrogen), transferred to nitrocellulose membrane, and analyzed using primary antibodies to GFP (1:1,000; Invitrogen), Dpm1p (1:500; Thermo Fisher Scientific), Pex3p (1:1,000; provided by R. Erdmann, Ruhr University, Bochum, Germany), and Porin (1:1,000; Invitrogen). Proteins were visualized using appropriate IRDye secondary antibody (LI-COR Biosciences; 1:10,000) followed by detection using the Odyssey system.
4
Protein Extraction and Quantification
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Proteins were extracted from cell pellets (corresponding to 1 OD600 unit) by NaOH treatment and boiling in sample buffer as described (Kushnirov, 2000 (link)). Equal amounts were loaded based on culture density (Appendix Fig S1B ) or protein quantification (Appendix Fig S7A ). For protein quantification, aliquots of each sample were acetone precipitated, followed by solubilization in urea/guanidine and protein determination using the Bradford assay as described (Sheen, 2016 (link)).
Proteins were analyzed using primary antibodies against Sct1p‐HA (anti‐HA, Thermo Scientific), Psd1p (kind gifts from Dr. Thomas Becker (Horvath et al, 2012 (link)), used in Appendix FigS1B , and from Dr. Steven Claypool (Onguka et al, 2015 (link)), used in Appendix Fig S7A ), Gpd1p (anti‐GAPDH, Thermo Fisher Scientific), and Dpm1p (Thermo Fisher Scientific). Proteins were visualized using horse radish peroxidase conjugated secondary antibodies (Bio‐Rad or Jackson Laboratories) or IRdye‐conjugated secondary antibodies (Li‐Cor Biosciences, Lincoln, NE).
Proteins were analyzed using primary antibodies against Sct1p‐HA (anti‐HA, Thermo Scientific), Psd1p (kind gifts from Dr. Thomas Becker (Horvath et al, 2012 (link)), used in Appendix Fig
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