Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Aspirate media from cultures wash cells with 1X PBS aspirate. Treat cells by adding fresh media containing regulator for desired time. Protein Blotting A general protocol for sample preparation. Detection Reagent: SignalFire™ ECL Reagent ( #6883).ī. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody ( #7074). Pore size 0.2 µm is generally recommended. Blotting Membrane and Paper: ( #12369) This protocol has been optimized for nitrocellulose membranes. Blue Prestained Protein Marker, Broad Range (11-250 kDa): ( #59329). Biotinylated Protein Ladder Detection Pack: ( #7727). Primary Antibody Dilution Buffer: 1X TBST with 5% BSA for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well. 10X Tris Buffered Saline with Tween ® 20 (TBST): ( #9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH 2O, mix. 
10X Tris-Glycine Transfer Buffer: ( #12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH 2O, mix.10X Tris-Glycine SDS Running Buffer: ( #4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH 2O, mix.
1X SDS Sample Buffer: Blue Loading Pack ( #7722) or Red Loading Pack ( #7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. 10X Tris Buffered Saline (TBS): ( #12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH 2O, mix. 20X Phosphate Buffered Saline (PBS): ( #9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2O, mix. NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. 
Solutions and Reagentsįrom sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit NOTE: Please refer to primary antibody product webpage for recommended antibody dilution. For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween ® 20 at 4☌ with gentle shaking, overnight.
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