Concentrate your supernatant from 200 ml down to 2 ml using Amicon Centrifugal Filters. You might then be able to see it. There are also plenty of published protocols for Salmonella OMVs isolation. Thanks

Hello,

Firstly, how are you disrupt the bacteria cells ? I dont see any information about it in your protocol. 

I don't know about OMVs in Salmonella specifically, but E. coli naturally does not produce many of OMVs, particularly during the log phase. The pellet is very hard to see, sometimes it is not visible at all; none the less, it is sufficient for a western blot analysis and I suppose the mass spec. Just resuspend in a small volume (50-100 uL) and you should be ok. 

See high resolution transmission electrom microscopy  after negativ stainig  by

phosphotungdting acid  pH-7.2.

Best Regards,

Karlen hovnanyan,PH>d.,D>sc.

Dear have u calibrated and validated ur centrifuge G - force requires calculation just donot go by rpm on display

Could u pls explain what was the cell load or did u diluted the sample as because if initial OS load is low then how do expect for good deposition

  I should get the pellet at the end of ultra-centrifugation. But I do not get the pellet. Sometimes I get a minute dot like pellet that is very less to do any type of analysis work.

Since u have written this as per ur work

So could u please enriche the sample if is in low density of recovery???

Concentrate your supernatant from 200 ml down to 2 ml using Amicon Centrifugal Filters. You might then be able to see it. There are also plenty of published protocols for Salmonella OMVs isolation. Thanks

There are several things you can do:

1. Increase the initial culture volume to 1L or more. More cells = more OMVs.

2. Grow culture to the appropriate phase. E. coli produce more OMVs in late stationary phase, however these things are highly species-specific so look at the literature first.

3. Induce OMV production. Normal growth medias may not induce large numbers of OMVs to be produced. Typical induction includes using iron-depleted media, application of heat and sub-lethal doses of antibiotics. However, as OMVs are produced as a response to cellular stress, you need to consider how this will affect potential OMV subpopulations (enrichment of lipids, selective packaging of proteins etc) and your downstream processes as your OMV yield will be related to those conditions.

4. As previously suggested by Abdi, you can concentrate your sample using a size exclusion centrifugal filter (amicon and sartorius both sell these). Your MW cut-off should be around 100kDa, allowing liquid and small bacterial proteins through, while retaining your OMVs in the retentate. So this acts both to concentrate and purify and appears to me at least, to becoming an essential step in OMV isolation protocols.

5. If using larger volumes as suggested (1L+) you won't be able to concentrate using centrifugal filters as the volume will be to big (they tend to take ~15mL at a time). For larger volumes you want to look at tangential flow concentrators which do the same thing (using a PES size-exclusion filter of 100kDa). These are available as disposable one-use units that are used in conjunction with a standard peristaltic pump, or as partially reusable units which can concentrate large volumes (5L at a time). However this can become expensive so plan your experiment according to your downstream needs.

6. You can precipitate the OMVs before ultracentrifugation using PEG8000, however i do not have experience with this and so cannot confidently advise further

7. Finally, if you have an invisible pellet (yes, this can be quite frustrating) or a pellet that is stuck to the tube very hard, remove the supernatant and add a small amount of your buffer (eg. 500uL PBS) to your tubes and allow to sit at room temperature for an hour or so. This helps resuspend the pellet fully and ensure that you're not losing the majority of your OMVs at the last step.

My conclusion:

Using greater initial volume and a concentration step (eg order of 10X), you should be able to visualise the pellet. In my experience with different centrifuge speeds and volumes, this pellet can vary in appearance. For E coli i find my pellets to be slightly orange tinged, jelly like stuff, loosely or detached, at the bottom of the centrifuge tube, meaning i have to carefully pipette out the supernatant manually rather than pouring off.

Also remember to filter through a 0.45uL PES filter before you put your final sample into an eppendorf tube to ensure to contamination upon storage (usually -80C, but avoid multiple freeze-thaw cycles). OMVs are also know to be stable at 4C, although exactly for how long I am not sure. I would suggest that the membrane proteins may start to degrade first, causing potential problems for downstream proteomics or functional assays.

Hope this helps!