Protein extracts from Drosophila larval brains were obtained by dissecting 10 larval brains in 0.7% NaCl and homogenizing them in 20 μl of 2X Laemmli buffer. Protein samples were loaded into a 4–20% Mini-PROTEAN TGX precast gel to perform electrophoresis (SDS-PAGE) and blotted using the Trans-Blot® Turbo™ Transfer System on a nitrocellulose membrane (Hybond ECL, Amersham). Filters were blocked in 5% non-fat dry milk dissolved in 0.1% Tween-20/PBS for 30 min at RT and, then, incubated with anti-Giotto (1:5000; Rabbit; ref. 60 (link)) and anti-γH2Av (1:1000; Mouse; Developmental Studies Hybridoma Bank, IA 52242) overnight at 4 °C. The membranes were then incubated with HRP-conjugated anti-Mouse and anti-Rabbit IgGs secondary antibody (1:5000; Amersham) for 1 h at RT and then washed again 3 times with 0.1%Tween-20/PBS. The chemiluminescent signal was revealed through either SuperSignal™ West Femto or SuperSignal™ West Pico substrate (Thermo Scientific™) using the ChemiDoc scanning system (Bio-Rad). Band intensities were quantified by densitometric analysis using the Image Lab 4.0.1 software (Bio-Rad). WB was repeated independently at least three times.
Chemidoc scanning system
Manufactured by Bio-Rad
Sourced in United Kingdom
The ChemiDoc scanning system is a versatile imaging platform designed for the detection and analysis of chemiluminescent, fluorescent, and colorimetric signals. It features a high-resolution camera and advanced optics to capture images of various samples, such as Western blots, gels, and membranes. The system provides researchers with accurate and sensitive imaging capabilities for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto, by Thermo Fisher Scientific (2 mentions)
Image lab 4, by Bio-Rad (2 mentions)
Anti γh2av, by Developmental Studies Hybridoma Bank (2 mentions)
Hrp conjugated anti mouse and anti rabbit iggs secondary antibody, by Cytiva (2 mentions)
Hybond ecl, by Cytiva (2 mentions)
Hybond, by Cytiva (2 mentions)
Supersignal west pico substrate, by Thermo Fisher Scientific (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Anti rabbit or anti mouse hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti ac tub, by Merck Group (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
3 protocols using chemidoc scanning system
1
Western Blot Analysis of Drosophila Larval Brain Proteins
2
Western Blot Analysis of Larval Testes
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To obtain testes extracts for the Western Blot analysis, larval testes were lysed in an ice-cold buffer containing 20 mM Hepes KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 420 mM NaCl, 30 mM NaF, 0.2 mM Na3Vo4, 25 mM β-glycerophosphate, 0.5 M PMSF, 0.1% NP40, 1× protease inhibitor cocktail (Roche, Basel, Switzerland). For immunoblotting, protein samples were resuspended in 1× Laemmli Buffer, run into SDS polyacrilammide gels, and electroblotted on a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) in a phosphate buffer containing 390 mM NaH2PO4 and 610 mM Na2HPO4. After blocking with 5% low-fat dry milk, the membrane was probed with appropriate primary antibody. Anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (1:5000; GE Healthcare, Chicago, IL, USA) were used as secondary antibodies. The blots were developed by the ECL or ECL Plus method (Amersham Biosciences, Little Chalfont, UK) and signals detected with the ChemiDoc scanning system (BioRad). The antibody dilutions were: Anti-DmATPCL (1:500), anti-acTub (Sigma; 1:10,000), anti Ac-Lys (1:1000; Merck-Millipore, Burlington, MA, USA).
3
Quantitative Western Blotting Analysis
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Protein extracts from Drosophila larval brains were obtained by dissecting 10 larval brains in 0.7% NaCl and homogenizing them in 20 µl of 2X Laemmli buffer. Protein samples were loaded into a 4-20% Mini-PROTEAN TGX precast gel to perform electrophoresis (SDS-PAGE) and blotted using the Trans-Blot® Turbo TM Transfer System on a nitrocellulose membrane (Hybond ECL, Amersham). Filters were blocked in 5% non-fat dry milk dissolved in 0.1% Tween-20/PBS for 30 min at RT and, then, incubated with anti-Giotto (1:5000; Rabbit; 59 ) and anti-γH2Av (1:1000; Mouse; Developmental Studies Hybridoma Bank, IA 52242) overnight at 4°C. The membranes were then incubated with HRPconjugated anti-Mouse and anti-Rabbit IgGs secondary antibody (1:5000; Amersham) for 1 hour at RT and then washed again 3 times with 0.1%Tween-20/PBS. The chemiluminescent signal was revealed through either SuperSignal TM West Femto or SuperSignal TM West Pico substrate (Thermo Scientific TM ) using the ChemiDoc scanning system (Bio-Rad). Band intensities were quantified by densitometric analysis using the Image Lab 4.0.1 software (Bio-Rad). WB was repeated independently at least three times.
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