Fat bodies from 5 larvae per sample were dissected in PBS and lysed directly in SDS sample buffer, with three or more biological replicates used for each experiment. Extracts were boiled 3 minutes, separated by polyacrylamide gel electrophoresis, and transferred to Immobilon-P membranes (Millipore, Billerica MA). Circulating Dilp3 levels were determined from hemolymph of 10 larvae per sample, diluted 1:100 in PBS and spotted (1 uL) onto methanol-soaked Immobilon-P membranes (Millipore, Billerica MA). Air-dried membranes were blocked in PBT + 5% BSA, and incubated overnight in blocking solution containing primary antibody. Signals were visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL) with BioMax Light (Kodak, Rochester NY) or HyBlot CL autoradiography film (Denville Scientific, Metuchen NJ), and quantitated using Adobe Photoshop software. Antibodies used were rabbit anti-phospho-T398 dS6K (1:250), rabbit anti-phospho-S505 dAkt (1:1,000), (both from Cell Signaling Technology, Beverly MA), rabbit anti-Dilp3 (1:1,000; gift of J. Veenstra, Université Bordeaux, Talence, France, ref. 34 (link)), and mouse anti-beta-tubulin E7 (1:1,000; Developmental Studies Hybridoma Bank, Iowa City IA).
Mouse anti beta tubulin e7
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
Mouse anti-beta-tubulin E7 is a monoclonal antibody that specifically recognizes the beta-tubulin protein. It is a useful tool for the detection and visualization of beta-tubulin in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Biomax light, by Kodak (2 mentions)
Rabbit anti phospho t398 ds6k, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho s505 dakt, by Cell Signaling Technology (2 mentions)
Hyblot cl autoradiography film, by Thomas Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Supersignal west pico plus chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Mouse anti hcn1, by Abcam (1 mentions)
4 protocols using mouse anti beta tubulin e7
1
Quantitative Western Blot Analysis of Insulin Signaling
2
Quantitative Western Blot Analysis of Insulin Signaling
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Fat bodies from 5 larvae per sample were dissected in PBS and lysed directly in SDS sample buffer, with three or more biological replicates used for each experiment. Extracts were boiled 3 minutes, separated by polyacrylamide gel electrophoresis, and transferred to Immobilon-P membranes (Millipore, Billerica MA). Circulating Dilp3 levels were determined from hemolymph of 10 larvae per sample, diluted 1:100 in PBS and spotted (1 uL) onto methanol-soaked Immobilon-P membranes (Millipore, Billerica MA). Air-dried membranes were blocked in PBT + 5% BSA, and incubated overnight in blocking solution containing primary antibody. Signals were visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL) with BioMax Light (Kodak, Rochester NY) or HyBlot CL autoradiography film (Denville Scientific, Metuchen NJ), and quantitated using Adobe Photoshop software. Antibodies used were rabbit anti-phospho-T398 dS6K (1:250), rabbit anti-phospho-S505 dAkt (1:1,000), (both from Cell Signaling Technology, Beverly MA), rabbit anti-Dilp3 (1:1,000; gift of J. Veenstra, Université Bordeaux, Talence, France, ref. 34 (link)), and mouse anti-beta-tubulin E7 (1:1,000; Developmental Studies Hybridoma Bank, Iowa City IA).
3
Western Blot Analysis of Trypanosome Proteins
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Cells were washed in Dulbecco’s phosphate buffered saline (DPBS) and boiled in 1x Laemmli sample buffer (Bio-rad). Samples were separated on a 10% acrylamide gel and transferred to nitrocellulose membrane. Membranes were blocked in phosphate buffered saline (PBS) + 5% milk and incubated overnight at 4°C in primary antibody diluted in blocking solution (1:3000 mouse anti-Ty BB2 (Bastin et al., 1996 (link)), 1:10,000 mouse anti-beta tubulin E7 (Developmental Studies Hybridoma Bank), or 1:10,000 rabbit anti-PFR2 (Saada et al., 2014 (link))). Membranes were washed three times in PBS + 0.05% Tween-20 (PBS-T) and incubated in goat anti-mouse or goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-rad) diluted 1:5000 in blocking solution. After washing three times in PBS-T, membranes were developed using the SuperSignal West Pico PLUS chemiluminescent substrate kit (Thermo) and images were captured on a Bio-rad ChemiDoc MP imaging system.
4
Protein Isolation from Mouse Cardiac Tissues
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For protein isolation, mouse SAN, atrial, and left ventricular tissue from 3-month-old female WT and HCN4FEA mice was snap-frozen in liquid nitrogen. Samples were homogenized on dry ice using a mortar and pestle and suspended in homogenization buffer (2% sodium dodecyl sulfate, 50 mM Tris and proteinase inhibitor cocktail mix). After heating at 95 °C for 15 min followed by centrifugation at 1000 × g for 10 min to remove cell debris, the resulting supernatant was used in western blot analysis as previously described38 (link). The following antibodies were used: mouse anti-HCN1 (1:1000; Abcam, UK), rat anti-HCN4 (1:500; Santa Cruz Biotechnology, USA), rabbit anti-HCN2 L (1:50046 (link)), and mouse anti-beta-tubulin (E7, 1:2000; Developmental Studies Hybridoma Bank, USA).
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