Fly heads or embryos were collected and lysed with protein extraction buffer (1% Triton X-100, 100 mM pH 6.8 Tris-HCl, 150 mM NaCl, and protease inhibitor tablet (Thermofisher Scientific A32955)). Protein samples were prepared with denaturing buffer (5% β-Mercaptoethanol, 0.2 M pH 6.8 Tris-HCl, 8% SDS, 40% Glycerol, 0.1% Bromophenol blue) and boiled at 95°C for 5 mins. Fifteen μg of sample was loaded in each well of a 7% SDS-PAGE gel, and then transferred to the membrane with Turbo Mini PVDF Transfer Pack (#1704156, Bio-Rad). The blot was then blocked with 5% BSA in TBST (0.5% Tween-20 in TBS), incubated with primary antibody at 4°C overnight, washed with TBST and incubated with secondary antibody (Jackson ImmunoResearch Inc.) at room temperature (RT) for 1 hour, and washed prior to ECL detection (20–302B, Genesee Scientific Corporation) and imaged using the ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Inc). Rabbit anti-DSCAM-cytoplasmic domain was used at 1:1500 (gift from Dr. Dietmar Schmucker) (Dascenco et al., 2015 (link)). Anti-alpha-tubulin was used at 1:400 (12G10, Developmental Studies Hybridoma Bank), rat anti-Elav at 1:750 (7E8A10, Developmental Studies Hybridoma Bank), and anti-actin at 1:100 (JLA20, Developmental Studies Hybridoma Bank).
Turbo mini pvdf transfer pack
Manufactured by Bio-Rad
The Turbo Mini PVDF Transfer Pack is a laboratory equipment designed for rapid protein transfer from polyacrylamide gels to PVDF membranes. It provides a convenient and efficient solution for Western blotting applications.
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2 protocols using turbo mini pvdf transfer pack
1
Drosophila Protein Expression Analysis
2
Drosophila Protein Expression Analysis
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Fly heads or embryos were collected and lysed with protein extraction buffer (1% Triton X-100, 100 mM pH 6.8 Tris-HCl, 150 mM NaCl, and protease inhibitor tablet (Thermofisher Scientific A32955)). Protein samples were prepared with denaturing buffer (5% β-Mercaptoethanol, 0.2 M pH 6.8 Tris-HCl, 8% SDS, 40% Glycerol, 0.1% Bromophenol blue) and boiled at 95°C for 5 mins. Fifteen μg of sample was loaded in each well of a 7% SDS-PAGE gel, and then transferred to the membrane with Turbo Mini PVDF Transfer Pack (#1704156, Bio-Rad). The blot was then blocked with 5% BSA in TBST (0.5% Tween-20 in TBS), incubated with primary antibody at 4°C overnight, washed with TBST and incubated with secondary antibody (Jackson ImmunoResearch Inc.) at room temperature (RT) for 1 hour, and washed prior to ECL detection (20–302B, Genesee Scientific Corporation) and imaged using the ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Inc). Rabbit anti-DSCAM-cytoplasmic domain was used at 1:1500 (gift from Dr. Dietmar Schmucker) (Dascenco et al., 2015 (link)). Anti-alpha-tubulin was used at 1:400 (12G10, Developmental Studies Hybridoma Bank), rat anti-Elav at 1:750 (7E8A10, Developmental Studies Hybridoma Bank), and anti-actin at 1:100 (JLA20, Developmental Studies Hybridoma Bank).
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