Sucrose gradient fractionation was performed as described previously (63). Cells were suspended in buffer S [25 mM tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% CHAPS, and protease inhibitor cocktail] and homogenized by 10 passages through a 25-gauge needle. After centrifugation at 10,000g for 30 min, soluble lysates were adjusted to a final concentration of sucrose (45%) and transferred to a 10-ml ultracentrifuge tube. Then, a discontinuous sucrose gradient was formed by sequentially layering 35% sucrose (3.2 ml) and 5% sucrose (3.2 ml), and the tubes were subjected to ultracentrifugation at 100,000g for 16 hours in Beckman SW 32.1 Ti rotor at 4°C with no brakes. Twelve 0.8-ml fractions were collected from the top to the bottom of the gradient, and the same volume of each fraction was analyzed by Western blotting.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.