Cells were grown in SD media to exponential phase (OD600 = 0.5–1), for cell fractionatiom74 (link). Spheroplasts were prepared by treatment with Zymolyase (Zymo Research; Orange, CA). After homogenization of spheroplasts by douncing in cold NMIB (0.6 M sorbitol, 5 mM MgCl2, 50 mM KCl, 100 mM KOAc, 20 mM Hepes pH 7.4) and centrifugation at 3000×g, the supernatant (Total fraction) was subjected to centrifugation at 10,170×g for 10 min, yielding a supernatant (Sup) and a mitochondrial enriched pellet fraction (Pellet). Subcellular fractions were assessed for Fzo1 (Anti-Myc tag: 1:1,000 (dilution), 9E10, Invitrogen, R950-25), cytosolic Pgk1 (Anti-Pgk1: 1:20,000 (dilution), Abcam, ab113687), and mitochondrial Por1 proteins (Anti-Por1: 1:1,000 (dilution), Abcam, ab110326). Whole scans of blots are shown in Supplementary Fig. 8 .
Ab110326
Manufactured by Abcam
Sourced in Canada
Ab110326 is a primary antibody for the detection of CHIP (C terminus of Hsp70-interacting protein) in various sample types. This antibody specifically recognizes the CHIP protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab113687, by Abcam (3 mentions)
Zymolyase, by Zymo Research (1 mentions)
Ab46765, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
G box, by Syngene (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Illustrator 2021, by Adobe (1 mentions)
Anti pgk1, by Abcam (1 mentions)
Anti vdac1 porin, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated horse anti mouse 7075, by Cell Signaling Technology (1 mentions)
Image lab software package, by Bio-Rad (1 mentions)
Anti cox 2, by Abcam (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Mitochondria isolation kit, by Merck Group (1 mentions)
5 protocols using ab110326
1
Cell Fractionation and Mitochondrial Enrichment
2
Immunoblot Analysis of SULT4A1 in Cell Lysates
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Equal amounts of yeast lysate fractions were resolved on 4–14% (4% stacking gel, 14% separating gel) Tris–Glycine SDS-PAGE gels and blotted onto a PVDF membrane (Bio-Rad) and immunostained with anti-SULT4A1 (12578-1-AP, Proteintech) first followed by stripping and staining with anti-Histone H3 (ab46765, Abcam), anti-GAPDH (GT239, GeneTex), or anti-VDAC1/porin (ab110326, Abcam) antibodies. Blots were visualized with Clarity Western ECL substrate (Bio-Rad) chemiluminescence and imaged using a gel-doc system (SYNGENE G:Box). All images were processed via Adobe Photoshop 2021 to correct signal levels of the complete image before cropping the shown area and placed into Adobe Illustrator 2021 to generate final figures. All samples shown in the figure panels were resolved in same gel.
3
Western Blot Analysis of Cellular Fractions
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Cell fractions and whole-cell lysates prepared as described above were electrophoresed on 8 to 10% SDS/PAGE and transferred to poly(vinylidene difluoride) membranes. Blots were incubated overnight in diluted primary antibody, probed with horseradish peroxidase (HRP)-conjugated horse anti-mouse (7075; Cell Signaling Technology) or goat anti-rabbit (7074; Cell Signaling Technology) secondary antibody and detected using Luminata Forte Western HRP Substrate (EMD Millipore). Blots were imaged with the Bio-Rad ChemiDoc XRS+ system; image processing was performed with the Image Lab software package (Bio-Rad). The primary antibodies and dilutions used were 1:1,000 anti-FLAG M2 (F1804; Sigma-Aldrich), 1:2,000 mouse anti-nuclear pore complex protein mAb 414 (obtained from Alexander Palazzo, University of Toronto, Toronto, ON, Canada), 1:1,000 anti-VDAC1/Porin (ab110326; Abcam), 1:1,000 anti-Cox2 (obtained from Tom Fox, Cornell University, Ithaca, NY), 1:2,500 anti-GAPDH (obtained from Cordula Enenkel, University of Toronto, Toronto, ON, Canada), 1:5,000 anti-Pgk1 (ab113687; Abcam), 1:2,000 anti-Killer toxin (obtained from Motomasa Tanaka, Tokyo Institute of Technology, Tokyo, Japan) (28 (link)), and 1:2,000 anti–L-A Gag (obtained from Reed Wickner).
4
Subcellular Distribution of PmOLV1 mRNA
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To determine the subcellular distribution of PmOLV1 mRNAs in fungal cells, an enriched mitochondria fraction was extracted using the mitochondria isolation kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s recommendation. Then, the protein concentration of supernatant and mitochondrial enriched precipitation fraction was quantified via bicinchoninic acid (BCA). The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocked with 5% dry milk in TBST (W/V), the membranes were immunoblotted with the two antibodies: cytosolic Pgk1 (Anti-PgK1, Abcam, ab113687) and mitochondrial Porin (Anti-Porin, Abcam, ab110326). Then, the membranes were further incubated with peroxidase conjugated secondary antibodies, followed by the protein bands detected using enhanced chemiluminescence HRP substrate and images taken using Chemiluminescence instrument.
5
Mitochondrial Protein Immunoblotting
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Primary antibodies used in this study include anti-Kar2 (SCBT sc-33630, 1:5000; RRID: AB_672118), anti-Cit163 (link) (custom made at Biomatik, 1:4000), anti-Tom7064 (link) (1:1000, a gift from Nora Vogtle, University of Freiburg), anti-Vdac (Abcam ab110326, 1:2000; RRID: AB_10865182); anti-GFP (SCBT sc-9996, 1:1000; RRID: AB_627695), anti-Sdh265 (link) (1:5000, a gift from Oleh Khalimonchuk, University of Nebraska). Secondary antibodies include goat anti-mouse (LI-COR 926-32210, 1:15000; RRID: AB_621842) and goat anti-rabbit (LI-COR 926-32211, 1:15000; RRID: AB_621843).
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