Protein concentration was determined using the bicinchoninic acid method. A total of 65 µl of each sample was mixed with 25 µl of NuPAGE LDS 4 × sample buffer (ThermoFisher Scientific, NP0007) and 10 µl of NuPAGE Sample Reducing Agent (ThermoFisher Scientific, NP0004) and heated at 70 °C for 10 min. Samples were run on 4–12% NuPAGE Bis–Tris gels (Invitrogen) and transferred to reinforced nitrocellulose membranes (Bio-Rad). After blocking with 5% fat-free milk in TBST buffer (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.6) for 60 min at room temperature, the membranes were incubated overnight with the following primary antibodies: rabbit anti-phospho-DRP1 (1:1000 dilution, Ser637, Cell Signaling, 4867), mouse anti-OPA1 (1:1000 dilution, BD Bioscience, 612,606), rabbit anti-FIS1 (1:500 dilution, FL-152, Santa Cruz, sc-98900), and mouse anti-VDAC1 (1:500 dilution, B-6, Santa Cruz, sc-390996). After washing, the membranes were incubated with peroxidase-labeled goat anti-rabbit IgG antibody (1:2000 dilution, Vector, PI-1000) or peroxidase-labeled horse anti-mouse IgG antibody (1:4000 dilution, Vector, PI-2000). Immunoreactive species were visualized using the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, 34,580) and an LAS 3000 cooled CCD camera (Fujifilm, Japan).
Las 3000 cooled ccd camera
Manufactured by Fujifilm
Sourced in Japan
The LAS 3000 is a cooled CCD camera designed for lab equipment. It features a high-resolution sensor and cooling technology to reduce thermal noise and improve image quality. The camera is capable of capturing detailed and sensitive images for various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Reinforced nitrocellulose membranes, by Bio-Rad (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer 4, by Thermo Fisher Scientific (2 mentions)
Peroxidase labeled goat anti rabbit igg antibody, by Vector Laboratories (2 mentions)
Peroxidase labeled horse anti mouse igg antibody, by Vector Laboratories (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Mouse anti opa1, by BD (1 mentions)
Taq dna polymerase, by Promega (1 mentions)
Reducing agent, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti caspase 3, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti actin, by Merck Group (1 mentions)
Reinforced nitrocellulose membranes, by Cytiva (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
6 protocols using las 3000 cooled ccd camera
1
Western Blot Analysis of Mitochondrial Proteins
2
Genotyping CHCHD4-Mutant Mice
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Genomic DNA was isolated from tail samples using DNA extraction kits (DNeasy, Cat. No. 69506, Qiagen, Hilden, Germany) according to the instructions of the manufacturer. The DNA concentration was measured by using a Nanodrop (Thermo Scientific, Waltham, MA, USA) and adjusted to around 50 ng/μl. The reaction mixture for genotyping contained 1 μl of genomic DNA, 0.2 mM dNTP, 4 μl 5 × green PCR buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2; Promega), 1 U of Taq DNA Polymerase (Promega, Madison, WL, USA), and either 0.5 μM of common and Wt primers or 0.5 μM of common and mutant primers. The PCR cycles were 95 °C for 30 s, 61°C for 30 s, and 72 °C for 45 s for 40 cycles. The following primers were used: IST11943B12-r common: 5′-GTGCTCCTCATAGGGATCATTGG-3′, Wt: IST11943B12-f 5′-TGGGCTGGTTAGTCAGTGATTGG-3′, and mutant: 5′-AAATGGCGTTACTTAAGCTAGCTTGC-3′. PCR products were separated on a 1.5% agarose gel containing SYBR green (1:10 000 dilution). A 100 basepair (bp) ladder was used to verify the sizes of the PCR products. The gels were imaged with a LAS 3000 cooled CCD camera (Fujifilm, Tokyo, Japan). CHCHD4-mutant mice were identified by the presence of a single 205 bp DNA band, and Wt mice were identified by a single 213 bp DNA band.
3
Western Blot Analysis of Actin and Caspase-3
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Homogenate samples with a volume of 65 μL were mixed with 25 μL NuPAGE LDS 4× sample buffer and 10 μL reducing agent (ThermoFisher Scientific) and heated at 70 °C for 10 min. Individual samples were run on 4–12% NuPAGE Bis-Tris gels (Novex) and transferred to reinforced nitrocellulose membranes. After blocking with 30 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1% Tween 20 containing 5% fat-free milk powder for 60 min at RT, the membranes were incubated with the rabbit anti-actin (1:200; Sigma) or rabbit anti-caspase3 (1:1000; Santa Cruz) primary antibodies at RT for 60 min. After washing, membranes were incubated with a peroxidase-labeled goat anti-rabbit secondary antibody (1:2000, Vector Laboratories) for 30 min at RT. Immunoreactive species were visualized using the Super Signal West Dura substrate (ThermoFisher Scientific) and an LAS 3000 cooled CCD camera (Fujifilm).
4
Western Blot Analysis of Apoptosis Markers
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The protein concentration was determined using the Pierce BCA kit (Perbio Science, Bonn, Germany). Samples were mixed with an equal volume of concentrated (3×) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) buffer and heated (96 °C) for 5 min. Pooled samples were run on 4–20% Tris–glycine gels (Novex, San Diego, CA, USA) and transferred to reinforced nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After blocking with 30 mM Tris–HCl (pH 7.5), 100 mM NaCl and 0.1% Tween 20 (TBS-T) containing 5% fat-free milk powder for 1 h at 25 °C, the membranes were incubated with primary antibodies: anti-caspase 3, anti-cleaved caspase 3, anti-PARP (1: 1000; Cell Signaling Technology, Danvers, USA). After washing, the membranes were incubated with appropriate secondary antibodies (Vector Laboratories, Burlingame, USA) for 30 min at 25 °C. Immunoreactive species were visualized using the Super Signal West Dura substrate (Pierce, Rockford, IL, USA) and a LAS 3000 cooled CCD camera (Fujifilm, Tokyo, Japan).
5
Parietal Cortex Protein Extraction and Western Blot
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Brain tissue from the parietal cortex in both hemispheres was dissected out and homogenized immediately on ice with a Dounce tissue homogenizer (Sigma, D8938) in tissue lysis buffer [15 mM Tris-HCl, pH 7.6, 320 mM sucrose, 1 mM dithiothreitol, 1 mM MgCl2, 3 mM EDTA-K, and 0.5% protease inhibitor cocktail (Sigma, P8340)]. The homogenate was centrifuged at 4°C and 9,200× g for 15 min, and the protein concentration of the supernatant was measured using the bicinchoninic acid method. Individual samples of 20 μg protein were loaded and run on 4%–12% NuPAGE Bis-Tris gels (Invitrogen, Cat# NP0336BOX) then transferred to reinforced nitrocellulose membranes (Bio-Rad, Cat# 162-0112). Membranes were incubated with mouse anti-fodrin (1:1,000 dilution, Enzo Life Sciences, Cat# BML-FG6090-0500) and rabbit anti-actin (1:200 dilution, Sigma, Cat# A2066) overnight at 4°C. After washing, the membranes were incubated with peroxidase-labeled goat anti-rabbit IgG antibody (1:2,000 dilution, Vector, Cat# PI-1000) or peroxidase-labeled horse anti-mouse IgG antibody (1:4,000 dilution, Vector, Cat# PI-2000). Immunoreactive species were visualized using the Super Signal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, Cat# 34580) and a LAS 3000 cooled CCD camera (Fujifilm, Tokyo, Japan).
6
Genotyping Atg7 Knockout Mice Using Cre and Flox Markers
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Floxed Atg7 mice were characterized previously15 (link) and were crossed with a Nes-Cre-driven line to produce Atg7flox/flox; Nes-Cre knockout (atg7 KO) and Atg7flox/+; Nes-Cre control mice (Ctrl). Genomic DNA was isolated from tail samples according to the manufacturer's instruction (Qiagen, 69506). The following primer sets were used for genotyping: for the Cre transgene (Cre-S:cre sense 55) 5′-TTT GCC TGC ATT ACC GGT CGA TGC AAC-3′ and (Cre-As:Cre AS1000) 5′-TGC CCC TGT TTC ACT ATC CAG GTT ACG GA-3′ and for the Ctrl and Atg7flox alleles (Hind-Fw) 5′-TGG CTG CTA CTT CTG CAA TGA TGT-3′ and (Pst-Rv) 5′-CAG GAC AGA GAC CAT CAG CTC CAC-3′. The reaction mixture for genotyping contained 1 μL of genomic DNA, 0.2 mM dNTP, 2.5 μL 10× PCR buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2, all from Sigma), 1 U of Taq DNA Polymerase (Sigma, D1806), and 0.5 μM of primer. The PCR parameters were 98°C for 20 s, 64°C for 30 s, and 72°C for 90 s for 30 cycles. PCR products were separated on a 1.5% agarose gel containing SYBR Green. A 100-base pair (bp) ladder was used to verify the size of the PCR products. The gels were imaged with an LAS 3000 cooled CCD camera (Fujifilm, Tokyo, Japan). atg7 KO mice were identified by the presence of both 500 bp and 1000 bp DNA bands. Ctrl mice were identified by the presence of 500 bp, 1000 bp, and 1500 bp products.
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