Althrough I have not done endosome isolation, but in my opinion, PNS can not be stored at -80, it should be loaded to discontineous centrifugation and procede at once. Think about if the freezing and thawing process may destroy cells, so the intact structure of PNS may be destroyed too.

I have seen protocols that freeze the PNS. But generally I agree with He Jianping. Go straight to next step until you ahe everything working. Here is a paper discussing the details. Although they are looking for specific Golgi vesicles, they characterize a continuous gradient method worth trying.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035695/

Your ultracentrifugation time needs to increased - maybe to 4hrs or so.

See also this discussion

https://www.researchgate.net/post/Endosomes_isolation

Thanks to both of you for your suggestions. I will go through the literature.

Based on my experience, it is essential to measure the refractive index of each sucrose concentration before making gradient.

It also depends on what kind of endosomes you are trying to isolate. Sucrose is OK but i would recommend Optiprep. Sucrose is very osmotically active and can rupture endosomal membrane. Optiprep does not have these concerns. Check the optiprep site for detailed methods. Another great way is to feed your cells paramagnetic particles (either in suspension or tagged to a ligand), break open the cells and use a magnet to pull out your endosomes.

Dear Neil,

I have to isolate both early and late endosoms. If we use paramagnetic particles or Dyanabeads, Is there no need of making sucrose or any other gradient?

it helps to clear things up. You can also use organelle specific antibodies hooked to magnetic beads such as Rab5 for early endosomes or Rab11 for late/recycling endosomes. Check our paper for details.

Mol Cell Biol 2009 20(8) 2337-2350

http://www.molbiolcell.org/content/20/8/2337.long

HI Sonam,

I also need to purify early endosome from  the mouse ES cell from our lab. However, i noticed that after we extract the early endosome from 25%-35% sucrose, we need to perform a methonal/choloride extraction. Do you know how to do the M/C extraction?

Im not sure what method you are using.  How much methanol are you using? Can you post details of the protocol. 

 Hi Lei,

The protocol  which i am following does not use methanol /chloride extraction .Instead  you can perform magnetic beads (Dynabeads) based purification of early or late endosomes after successfully isolating through ultracentrifugation.

I don't know about your protocol, your protocol might be using methnol extraction.

I noticed Lei Hong's ,ethod uses methanol, but yours does not.  You cannot freeze the samples midway through the protocol as this will cause breakage of the vesicles and leakage of contents.  Purified samples can be frozen. The PNS step is right at the beginning, so you need to carry on with the prep.  You can measure the refractive index once, but it is not really necessary to do it each time, it will not vary that much.  Good luck

Hi Sonam,

I have the same problem as you now. After i centrifuge my sample (Firstly I loaded the centrifuge tube with PNS adjusted to 40% with 62% sucrose solution. Then overlaid with 1.5 ml of 35%, then 1ml of 25% and finally fill the tube with HB buffer). I DID NOT observe any interphase. Can you detect the interphsase now? According to the ptotocl, i should see a milky band in different interphase.

Also, after you recover the early endosome from 25% -35%, do yo diresctly load the sample on SDS page withour any precipitation???

Thank you!

Lei

Make sure that you are using enough starting material.  Also when you remove the interface you can dilute in homogenization buffer and pellet at 100,000g and solubilize the pellet in SDS.

HI Neil

 I saw an interphase at 35% sucrose. however, the 25% sucroser interphase can not be detected. 

You need a lot of cells to have enough protein to detect.  think about how much you are diluting this!

I have used the following protocol for endosomal isolation. It works well.

https://www.ncbi.nlm.nih.gov/pubmed/10485263

Hello,

I'm also a newbie trying to isolate endolysosomal fragments and I was thinking of giving a shot to the protocol Takafumi Hasegawa attached.

I'm a bit puzzled on how to make the homogenization buffer in the protocol you attached: 10 mM tris(hydroxymethyl)aminomethane/acetic acid pH 7.0, 250 mM sucrose. Is there a percentage for acetic acid or it is just used to adjust the pH?

Thanks,

Ioanna