Protein lysates of cells and tissue were prepared using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, 2 mM EDTA) complemented with protease and phosphatase inhibitors (Roche) and protein concentration was quantified using the BCA protein assay kit (Pierce). Protein lysate was loaded onto NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen) and transferred onto Trans-Blot® Turbo™ Mini or Midi Nitrocellulose membranes (BioRad) using Trans-Blot Turbo Transfer System (BioRad). Membranes were blocked in 10% Western Blot Blocking Reagent (Roche) or 3% BSA for 1 h at room temperature (RT). Primary antibody incubation was performed overnight at 4°C. Membranes were washed using TBS-T and subjected to secondary fluorochrome-conjugated antibodies for 1 h at RT and protein was detected using the Odyssey CLx imaging system and processed using ImageJ software 1.48v. Antibodies are listed in Supplemental Table 1.
Western blot blocking reagent
Manufactured by Roche
The Western Blot Blocking Reagent is a laboratory product used to block non-specific binding sites on a membrane during the Western blotting process. It helps to reduce background noise and improve the specificity of antibody detection.
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Lab products found in correlation
3 protocols using western blot blocking reagent
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Western Blotting of Cellular Proteins
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Western Blotting of Cellular Proteins
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In Situ Hybridization and Immunostaining Protocol
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Colorimetric (NBT/BCIP) or fluorescence in situ hybridizations were performed as described (Lander and Petersen, 2016) after fixation in 4% formaldehyde and bleaching (Pearson et al., 2009) using blocking solution containing 10% horse serum and western blot blocking reagent (Roche) (King and Newmark, 2013) . Digoxigenin-or fluorescein-labeled riboprobes were synthesized as described (Pearson et al., 2009) and detected with anti-digoxigenin-HRP (1:2000, Roche/Sigma-Aldrich 11207733910, lot 10520200 anti-fluorescein-HRP (1:2000, Roche/Sigma-Aldrich 11426346910, lot 11211620) or anti-digoxigenin-AP (1:4000, Roche/Sigma-Aldrich 11093274910, lot 11265026). Hoechst 33342 (Invitrogen) was used at 1:1000 as a counterstain.
For immunostainings, animals were fixed in Carnoy's solution as described (Hill and Petersen, 2015) , using tyramide amplification to detect labeling with rabbit anti-6G10 (1:3000, Cell Signaling D2C8, lot 3377S).
For immunostainings, animals were fixed in Carnoy's solution as described (Hill and Petersen, 2015) , using tyramide amplification to detect labeling with rabbit anti-6G10 (1:3000, Cell Signaling D2C8, lot 3377S).
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