I am trying to isolate polysomes using sucrose density gradient system from cancer cell line. I have to collect 13 different fractions after centrifuge but we don't have any fractionation system to collect these fractions.
Have any alternate options to collect different fraction.....please tell me
We have always collected the fractions inserting a glass capillary slowly and delicately through the gradient down to the bottom of the tube. The capillary is mounted on one end of a thin rubber tube that goes through a peristaltic pump. From the other end we manually collect 1 ml fractions (of course any volume can be collected). The flow has to be slow, max 1 ml/min so not to disrupt the gradient while sucking. The capillary and the tubing are quite thin (0.5 mm), minimizing dead volume (maybe 1 ml) and mixing. If you have a cell spectrophotometer to which connect your tube you can read continuously the polysomal rRNA content and create a graph. But if you don't have it, you can always extract and precipitate the RNA from each fraction and run an Ethidium Bromide gel (to see the rRNA profile) or a Northern blot or RT-(q)PCR.
use sucrose density gradient and for recovery of your sample Use a needle to puncture tube 3-5 mm above your desired organelle, and let the medium flow out of the tube.
read following article for more details about different organelle fractionation.
We have always collected the fractions inserting a glass capillary slowly and delicately through the gradient down to the bottom of the tube. The capillary is mounted on one end of a thin rubber tube that goes through a peristaltic pump. From the other end we manually collect 1 ml fractions (of course any volume can be collected). The flow has to be slow, max 1 ml/min so not to disrupt the gradient while sucking. The capillary and the tubing are quite thin (0.5 mm), minimizing dead volume (maybe 1 ml) and mixing. If you have a cell spectrophotometer to which connect your tube you can read continuously the polysomal rRNA content and create a graph. But if you don't have it, you can always extract and precipitate the RNA from each fraction and run an Ethidium Bromide gel (to see the rRNA profile) or a Northern blot or RT-(q)PCR.
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