Mitochondrial and cytosolic fractions were generated by homogenizing freshly excised hearts in homogenization buffer (20 mM HEPES, 140 mM KCl, 10 mM EDTA, 5 mM MgCl2, pH 7.4) with a Dounce tissue homogenizer, centrifuging the homogenate at 800 × g for 10 minutes and centrifuging the resulting supernatant at 8,000 × g for 10 minutes [77 (link)]. The supernatant is the cytosolic fraction. The pellet was washed by centrifugation at 10,000 × g and represents the mitochondrial fraction. Whole-cell extracts and mitochondrial membranes were prepared as described previously. Samples were loaded on SDS-PAGE, transferred to nitrocellulose or PVDF membranes, and incubated with specific antibodies [78 (link)]. Bands were visualized using horseradish peroxidase–conjugated secondary antibodies and the ECL detection system (GE Healthcare, Piscataway, NJ) or fluorophore-conjugated secondary antibodies and the Odyssey fluorescence detection system (Li-Cor Biosciences, Alpharetta, GA).
Odyssey fluorescence detection system
Manufactured by LI COR
Sourced in United Kingdom
The Odyssey fluorescence detection system is a versatile and advanced imaging platform designed for a wide range of applications in life science research. It utilizes near-infrared fluorescence technology to enable high-sensitivity and quantitative detection of proteins, nucleic acids, and other biomolecules. The system is capable of acquiring high-resolution, digital images and performing quantitative analysis, making it a valuable tool for researchers in various fields.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection system, by GE Healthcare (1 mentions)
Anti 14 3 3 antibody, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Image studio, by LI COR (1 mentions)
Anti α tubulin antibody, by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Nitrocellulose, by Cytiva (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Cleaved caspases 3, by Cell Signaling Technology (1 mentions)
B cell lymphoma extra large bcl xl, by Cell Signaling Technology (1 mentions)
Poly adp ribose polymerase 1 parp 1, by Santa Cruz Biotechnology (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab2231, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using odyssey fluorescence detection system
1
Mitochondrial and Cytosolic Fractionation
2
Western Blot Analysis of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins (5–10 μg) from cell/tissue lysates or homogenates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% BSA or skim milk powder in Tris-buffered saline containing 0.1% (v/v) Tween 20 for 1 hr, followed by an overnight incubation at 4°C with specific primary antibody solutions. Membranes were incubated with an appropriate secondary antibody for 1 hr before signals were detected using ECL (Thermo Fisher Scientific or Millipore) on the Chemidoc MP (Bio-Rad) or on the Odyssey Fluorescence Detection System (LiCOR). Antibodies detecting multiple mitochondrial complex subunits (Cat. No. 45–8099) and PRDX1 were from Thermo Fisher Scientific (Cat. No. PA3-750), anti-PRDX2 (Cat. No. ab109367, clone: EPR5154) and anti-catalase (Cat. No. ab52477) antibodies were from Abcam, anti-PRDX3 antibody from Ab Frontier (Cat. No. LF-PA0030), anti-14-3-3 antibody was from Santa Cruz (Cat. No. sc-629, clone K19), anti-pT642 TBC1D4 (Cat. No. 4288), anti- TBC1D4 (Cat. No. 2670), anti-pT308 Akt (Cat. No. 9275), anti-pS473 Akt (Cat. No. 4051) and anti-Akt (Cat. No. 4685) antibodies were from Cell Signaling Technologies and anti-α-tubulin antibody was from Sigma Aldrich (Cat. No. T9026). Antibody to GLUT4 was generated in-house. Densitometry analysis was performed using Image Lab 5.2.1 (Bio-Rad) or Image Studio (LiCOR).
3
Immunoblotting of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For preparation of whole-cell lysates, cells were washed three times with phosphate-buffered saline (PBS) and resuspended in 2× Laemmli buffer. After heating to 95°C for 5 min, proteins were resolved by SDS–PAGE and transferred to nitrocellulose (Whatman). Immunoblots were blocked with 5% wt/vol milk in Triton X-100 in PBS and then probed with primary antibodies (anti-INCENP, anti-AIM, or DM1α to detect tubulin) at 1:500 for 1 h. Blots were then probed with species-appropriate fluorescently tagged secondary antibodies for 45 min. Fluorescence was measured using an Odyssey fluorescence detection system (LI-COR Biosciences).
4
Western Blotting of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates were prepared by washing cells three times with Dulbecco's PBS (DPBS), suspending them in 2× Laemmli buffer, and boiling them for 5 min. Mitotic lysates were prepared by synchronizing cells with a thymidine block; 10 h after thymidine washout, cells were arrested in mitosis for 3 h in 10 μM STLC; the plates were rinsed three times in DPBS; and mitotic cells were collected by shake-off, lysed in 2× Laemmli buffer, and boiled as described. Proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes. Membranes were blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE), probed with primary antibodies diluted in 5% (wt/vol) milk in PBS–Tween-20 (PBST) for 1 h, and then probed with species-appropriate fluorescently tagged secondary antibodies in PBST plus milk for 45 min. Fluorescence was measured with an Odyssey fluorescence detection system (LI-COR Biosciences).
5
Western Blot Analysis of SRRM4 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysate was prepared in RIPA buffer according to the manufacture’s instruction (Nacalai). Total protein (50 μg) was fractionated by SDS-PAGE on a 7% Protean TGX gel and transferred to nitrocellulose membrane using Transblot Turbo (Bio-Rad). Membranes were blocked with TBS (20 mM Tris-buffered saline, pH7.2) with 1.0% skim milk at room temperature (RT) and then reacted with appropriate primary antibody in TBS containing 0.1% Tween 20 (TBS-T) at 4 °C for overnight (−15 hr). After washing with TBS-T, a fluorescent-labeled secondary antibody (dilution of 1/15,000 or 20,000) was added and incubated for 1 hour at room temperature. Protein bands were visualized using an Odyssey fluorescence detection system (LI-COR). Antibodies (anti-SRRM4, abcam) were obtained from Santa Cruz Biotechnology, and an anti-β-actin antibody was from Wako Chem.
6
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer supplemented with protease inhibitor. Protein (20-40 μg) was resolved on a 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis gel and transferred to the PVDF membrane. The membrane was blocked with 3% to 5% bovine serum albumin and probed by the primary antibodies against poly-(ADP-ribose) polymerase-1 (PARP-1) (Santa Cruz Biotechnology), B-cell lymphoma-extra large (BCL-XL) (Cell Signaling Technology), cleaved caspases-3 (Cell Signaling Technology), and GOLM1 (Sino biology). The data were acquired by Odyssey fluorescence detection system (LI-COR Biosciences, Lincoln, NE).
7
Western Blot Analysis of DDAH-1 in Rat Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Urethane-anesthetized rats were killed by decapitation and tissues (spinal dorsal horn, DRG, and hippocampus) were dissected and snap frozen. Tissues were homogenized in RIPA (radioimmunoprecipitation assay) buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS + 0.5% deoxycholic acid + complete protease inhibitor cocktail) using a glass homogenizer. Homogenates were centrifuged at 14,000 rpm for 10 minutes at 4°C, and supernatants containing whole-cell tissue lysates were collected. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Cramlington, United Kingdom). Laemmli loading buffer was added to protein lysates (40 μg) and samples were incubated at 70°C for 30 minutes, before loading onto 8% gels, and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Proteins were transferred to nitrocellulose membranes and probed overnight at 4°C with goat anti-DDAH-1 (1:500; ab2231; Abcam, Cambridge, United Kingdom) or rabbit anti-neuronal β-III Tubulin (1:3000; ab18207; Abcam), which served as a loading control. Membranes were incubated with IRDye-linked donkey anti-goat 680 or donkey anti-rabbit 800CW secondary antibody (1:15,000) for 1 hour at RT. Proteins were revealed using the Odyssey fluorescence detection system (Licor, Cambridge, United Kingdom).
Proteins were transferred to nitrocellulose membranes and probed overnight at 4°C with goat anti-DDAH-1 (1:500; ab2231; Abcam, Cambridge, United Kingdom) or rabbit anti-neuronal β-III Tubulin (1:3000; ab18207; Abcam), which served as a loading control. Membranes were incubated with IRDye-linked donkey anti-goat 680 or donkey anti-rabbit 800CW secondary antibody (1:15,000) for 1 hour at RT. Proteins were revealed using the Odyssey fluorescence detection system (Licor, Cambridge, United Kingdom).
8
Investigating BMDM C/EBPα Regulation
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!