Running a sucrose gradient - (Apr/29/2012 )

Hi guys, I'm from a lab heavily focused on animal behaviour so our collective knowledge on molecular techniques is somewhat limited!

I've got a few questions regarding sucrose gradients and planning an experiment. I'd like to isolate our drug (tritiated and biotinylated forms) bound to its receptor/s. I'm trying to mimic some experiments run back in the 70's (Goldstein A, Lowney LI & Pal BK (1971) Stereospecific and nonspecific interactions of the morphine congener levorphanol in subcellular fractions of mouse brain. Proc. Natl. Acad. Sci. U.S.A. 68: 1742–1747.)

The details for the previous experiments -
Used a 0.8 (27%) – 1.75M (60%) 24ml sucrose gradient 500µL fractions. (2ml of homogenate layered on top). Centrifuged for 180 mins at 50000 x g in the SW25 rotor (k-factor of 112.6).

My questions are as follows:
1: Does 0.8 - 1.75M sucrose gradient mean there are only 2 gradients or multiple gradients in between?

2: We will be using the SW 28 rotor so using the rotor k-factor (k-factor of 245.5) i altered the time/speed to 16652RPM (50000 x g) for 205 mins. Does this seem like an acceptable time/force to use?

3: How much protein can i run per ml of sucrose? i may have to downsize from 24ml to 5ml due to the tubes i have available.

4: Will polytron homogenisation of the protein suffice or should i also lyse?

Thanks, Jacob

1: the way you write it would indicate a linear gradient of sucrose (check the reference for specifics).

2: if it uses the same tubes then you should be able to spin for the same time at the same g force(max). spinning a little extra shouldn't hurt especially if your fraction of interest falls somewhere around the middle of the gradient.

3: this gets a little more complicated, the gradient is 24ml, sample 2 ml and you leave a small gap to the top of the tube (~1-2mm, prevents spillage during manipulation). if your tubes hold 5ml then you have to scale everything down proportionately, and still leave a gap on top. you should measure the volume that gives you a proper gap.

4: polytron should be okay but i prefer a tissue grinder (see my response to your other topic). you get a finer homogenization.

i just checked the paper, they use a loose fitting homogenizer (tissue grinder). they also used a dounce with the "A" pestle (0.1mm clearance, check the clearance in the data sheet with the homogenizer, some brands reverse the "A" and "B" designations). based on the paper, polytron probably won't suffice.