That is higher protein concentration than I get with exosome isolation from 40mL conditioned cell culture supernatant, so does not seem very low to me (I get around 200ug/mL). How much media are you collecting from? If your DLS and TEM are conclusive you have particles in the size range 20nm-140nm and resemble exosomes on TEM, then perhaps it is your antibodies for those markers, do you have a CD63 Ab?

Thank you for your reply Mr. Phillip

i use about 100ml of condition cell culture media. first i let the cell culture media to grow for around 80 - 90 % confluence. then starve of 24hrs -30 hrs in RPMI with 1 % Antibiotics and  without FBS.Then collect the all  media and centrifuge in 300 g for 3mins. then 2500g for 15mins collect the supernatant . Then centrifuge for 15mins in 10000g then take the supernatant and filter by .22um . then again centrifuge for 100000g for 2hrs in fixed rotor. and then throw the supernatant and wash the pellet  by 1 ml of PBS FOR 100000G FOR 1 hrs. After than pour off the pbs and mix then exosome pellet in 100ul of PBS . THis is the  process i use for ultracentrifugation process. And for protein quantification i use Bradford assay. And directly lyse the exosome 100 ul pbs sample by 11 ul of 10x ripa buffer and i also use 1ul of protease inhibitor.And  use 4xreducing loading buffer and incubate for 30 mins with vortex in 10mins of interval.And the lysate is ready for western blotting. Previous i used only 9 ug for western blotting . For next western blotting i will be using 15 ug in each well.And i have CD 63 ab too. i will use that antibody.

Can you suggest me anything by looking my above procedure? Is there any mistake in my process?How much ug do you load to get the bands in western blotting?

Hello Sumeet.

There are at-least two steps where you are losing your exosomes. First of all, following incubation, please collect your exosomes as follows:

1. centrifuge at 500 x g for 10 min. Removes cells and debris.

2. centrifuge at 10, 000 x g for 30 min. Removes apoptotic bodies.

3. centrifuge at 50, 0000 x g for 60 min. Removes micro vesicles.

4. centrifuge at 100, 000 x g for 120 min. You get true exosomes.

Please do not pass through 0.2 um membrane filter for as much as 50% exosomes are lost. If you must do this step for some particular reason, use 0.45 um pre-washed membrane filter.

After final centrifugation, have your tubes(s) stand inverted on tissue paper for 5 min. Add appropriate volume of PBS that covers the exosome pellet. Let exosomes go into buffer on their own by incubating the tube(s) at 4 C overnight. Please do now wrestle with exosomes by pipeting up and down. By doing so you lose more exosomes than you get into solution. If you must wash exosomes, add sufficient PBS (usually equal to the starting sample volume), centrifuge at 100,000 x g for 120 min and repeat the process mentioned above.

Regards.

 Thank you Syed A Ali.I will follow that.I have a question how much PBS buffer should i use for the collection of exosome? And do exosome effected by stress of pipeting  .Previously i used 100ul of pbs for 100ml of cell culture media for collection of exosome pellet. And what lysis buffer do u use for lysis.  i use 10x ripa buffer 11ul for 100ul of PBS and incubate for 30 mins. and use reducing loading buffer for loading in western blotting.Can u suggest me? i have not get the bands in western blotting

Our ultracentrifuge rotor accomodates 25 mL bottles to which I add 22 mL of sample. I mark the orientation and add 100 uL of PBS. For SDS PAGE/Western blot analysis, we do not treat sample with RIPA but directly dilute them in 5X loading dye (from Thermo Pierce). I do not know why you are not getting your bands lighted up for exosome markers (we use anti CD63, CD81, and Rab5). Have you checked if your antibodies are working fine? 

Whether exosomes get affected by pipeting stress I do not know. However, extensive pipeting lead to foaming and small particles such as exosomes (also true for viruses) begin to float on foam and not get collected.

I think the 5xloading dye is reducing dye. Do you heat the sample for five minutes before loading? and do you use protease inhibitor.I use 1ulAnd do you do protien quatificaion for loading in westrn blotting.  And is my cell confluence % and starvation time correct as i have explained above?And which company primary antibodies do u use? I have bioscience antibody and abcam antibody. i have only used bioscience antibody . Thanks for your reply

Two tips from our experience:

Run non-reducing gels when blotting for tetraspanins (e.g CD9, CD63). It vastly improves the efficiency of detection.

I don't think you mentioned the source of your exosomes. We tried many Ab raised against human CD9 and CD63 for the analysis of murine exosomes. None of them recognized the mouse proteins.

Thank you for your reply Professor Pamela Wearsch ,

I was using only using CD9 antibodies. For next experiment I will use non-reducing gels.I am using A549 cell line. I use ultracentrifugation process as I have already mentioned above.I use about 100ml of Culture media  to gain 100ul exosome sample.I use Stericup filter .  I do bardford assay for qualification of protein and its very low about 600ug/ml. Can filter be one of the main reason for low exosome yield.( Stericup Filter unit)? And I do a lot of pipetting and vortex to  detach the exosome pellet from the UC tube by PBS.Can you give me some suggestion Professor.

Dear Syed A Ali,

Are you using swinging bucket rotor? How do you mark the orientation of the tubes to know where is the pellet? And do you see any pellet formation when working with cell culture media (in the last centrifugation)?

Hi everyone!

I know that this is already from a few years ago but I'm having a similar problem. I want to do BCA assay to quantify exosomal proteins to do WB. But I haven't found a paper that describes exosomal lysis for protein quantification. Did you manage to solve your problem? Is it posible to lyse the exosomes with a lower sample volume? I tried on NanoDrop (to try to save some exosomes) but the results are weird, thus I need to find another viable way.

Thank you for the attention!

Syed A Ali

Sir in step 3 is this 50, 0000 x g for 60 min or 50000 x g for 60 min?