S. pombe whole cell extracts were prepared by bead beating using a FastPrep-24 machine (MP Biomedicals) [30 (link), 31 (link)]. Proteins were separated by 15% SDS/PAGE and analyzed by immunoblotting using anti-Hsp3101 antibody (1:5000), anti-c-Myc antibody (1:5000) to detect Hsp3102-Myc, Hsp3103-Myc and Hsp3104-Myc, anti-Sdj1 antibody (1:5000) and anti-Sla1 antibody (1:5000) as a loading control. IRDye 800CW conjugated goat anti-rabbit IgG (LI-COR Biosciences) was used as secondary antibody. The fluorescent bands were detected using an Odyssey near-infrared fluorescence scanner (LI-COR Biosciences).
Odyssey near infrared fluorescence scanner
Manufactured by LI COR
Sourced in Niger
The Odyssey near-infrared fluorescence scanner is a lab equipment product designed for detecting and quantifying fluorescent signals. It utilizes near-infrared technology to capture and analyze data from a variety of sample types. The core function of the Odyssey scanner is to provide high-sensitivity detection and imaging capabilities for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Fluorophore conjugated anti rabbit or anti goat iggs, by LI COR (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Irdye 800cw conjugated goat anti rabbit igg, by LI COR (1 mentions)
Fastprep 24 machine, by MP Biomedicals (1 mentions)
Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Hybond ecl transfer membrane, by GE Healthcare (1 mentions)
Anti gst, by GenScript (1 mentions)
Hybond, by Cytiva (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Affinity Biosciences (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Anti myc, by GenScript (1 mentions)
5 protocols using odyssey near infrared fluorescence scanner
1
Immunoblotting of S. pombe Whole Cell Extracts
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S. pombe whole cell extracts were prepared by bead beating using a FastPrep-24 machine (MP Biomedicals) [30 (link), 31 (link)]. Proteins were separated by 15% SDS/PAGE and analyzed by immunoblotting using anti-Hsp3101 antibody (1:5000), anti-c-Myc antibody (1:5000) to detect Hsp3102-Myc, Hsp3103-Myc and Hsp3104-Myc, anti-Sdj1 antibody (1:5000) and anti-Sla1 antibody (1:5000) as a loading control. IRDye 800CW conjugated goat anti-rabbit IgG (LI-COR Biosciences) was used as secondary antibody. The fluorescent bands were detected using an Odyssey near-infrared fluorescence scanner (LI-COR Biosciences).
+ Expand
S. pombe whole cell extracts were prepared by bead beating using a FastPrep-24 machine (MP Biomedicals) [30 (link), 31 (link)]. Proteins were separated by 15% SDS/PAGE and analyzed by immunoblotting using anti-Hsp3101 antibody (1:5000), anti-c-Myc antibody (1:5000) to detect Hsp3102-Myc, Hsp3103-Myc and Hsp3104-Myc, anti-Sdj1 antibody (1:5000) and anti-Sla1 antibody (1:5000) as a loading control. IRDye 800CW conjugated goat anti-rabbit IgG (LI-COR Biosciences) was used as secondary antibody. The fluorescent bands were detected using an Odyssey near-infrared fluorescence scanner (LI-COR Biosciences).
2
Protein Immunoblotting Workflow
3
Comprehensive Mitochondrial Protein Analysis
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S. pombe whole cell extracts were prepared by alkaline extraction (58 (link)) or by breaking cells with glass beads using a FastPre-24 bead beater (MP Biomedicals) (59 (link)). Mitochondrial protein extracts were prepared as described above. Proteins were resolved by electrophoresis on SDS-PAGE, and the separated protein bands were transferred electrophoretically to a Hybond ECL transfer membrane (GE healthcare). Blots were probed with anti-Cox1 (1:1000), anti-Cox2 (1:1000), anti-Cox3 (1:400), anti-Cox4 (1:1000) and anti-Cob1 (1:500), anti-Trz2 (1:1000), anti-Sla1 (1:5000), anti-HSP60 (1:1000; Sangon Biotech), anti-FLAG (1:000; Sigma), anti-CBP (1:1000; GenScript), anti-HA (1:2000; Sigma-Aldrich), anti-c-Myc (1:2000; Affinity Biotech), anti-His (1:2000; GenScript) and anti-GST (1:2000; GenScript) Abs. Secondary Abs used were IRDye 800CW conjugated goat anti-rabbit or anti-mouse Abs (LI-COR Biosciences). Bands were detected using an Odyssey near-infrared fluorescence scanner (LI-COR Biosciences).
+ Expand
S. pombe whole cell extracts were prepared by alkaline extraction (58 (link)) or by breaking cells with glass beads using a FastPre-24 bead beater (MP Biomedicals) (59 (link)). Mitochondrial protein extracts were prepared as described above. Proteins were resolved by electrophoresis on SDS-PAGE, and the separated protein bands were transferred electrophoretically to a Hybond ECL transfer membrane (GE healthcare). Blots were probed with anti-Cox1 (1:1000), anti-Cox2 (1:1000), anti-Cox3 (1:400), anti-Cox4 (1:1000) and anti-Cob1 (1:500), anti-Trz2 (1:1000), anti-Sla1 (1:5000), anti-HSP60 (1:1000; Sangon Biotech), anti-FLAG (1:000; Sigma), anti-CBP (1:1000; GenScript), anti-HA (1:2000; Sigma-Aldrich), anti-c-Myc (1:2000; Affinity Biotech), anti-His (1:2000; GenScript) and anti-GST (1:2000; GenScript) Abs. Secondary Abs used were IRDye 800CW conjugated goat anti-rabbit or anti-mouse Abs (LI-COR Biosciences). Bands were detected using an Odyssey near-infrared fluorescence scanner (LI-COR Biosciences).
4
Western Blot Protein Detection
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Cell or tumor lysates were fractioned on NUPAGE 4–12% Bis–Tris gels (Thermo Fisher Scientific) and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE), blotted with the primary antibodies as described in Supplementary Table 1 , and visualized using fluorophore-conjugated anti-rabbit or anti-goat IgGs (LI-COR) (1:5000 dilution) on an Odyssey near-infrared fluorescence scanner (LI-COR). The original un-cut western blot images are included in Supplementary Figure 15 .
5
Western Blot Analysis of Mitochondrial Proteins
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Proteins were separated using SDS/PAGE and transferred to nitrocellulose membranes (GE Healthcare Life Science). Anti-peptide Abs against Cox1, Cox2, Cox3, Cob1, and Atp6 were prepared as described previously (21 (link)). Anti-peptide Abs against Mrp5, Rsm24, Mrpl16, and Mrpl40 were generated as described (55 (link)). Other primary Abs used are anti-HA (Cat. No. T0008, Affinity Biosciences), anti-FLAG (Cat. No. T0003, Affinity Biosciences), anti-CBP (Cat. No. A00635, Genscript Biotech Corporation), anti-Myc (Cat. No. T0001, Affinity Biosciences), and anti-HSPD1 (Cat. No. D120788, Sangon Biotech) for detection of Mcp60. The secondary Abs were IRDye 800CW goat anti-rabbit Ab (Cat. No. 926-32211, LI-COR Biosciences) and IRDye 800CW goat anti-mouse Ab (Cat. No. 926-32210, LI-COR Biosciences). Bands were detected using an Odyssey near-infrared fluorescence scanner (LI-COR Biosciences), and quantified using Image Studio Lite software (LI-COR Biosciences).
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