Dear all,
I am currently doing research on extracellular vesicles (EVs). I'd like to improve the isolation method by trying out size-exclusion chromatography (SEC) compared to serial ultracentrifugation. However, the amount of starting medium we are dealing with is quite big. We have around 200ml conditioned cell medium.
Therefore, I'd like to ask if anyone knows how we can concentrate our starting medium in order to add it on the SEC-columns without the need of a new machine?
Thank you already in advance,
Vera
From experience in our lab, SEC is no good for isolation. However, it is good for purification. My advice would be to isolate using gentrification methods and place your isolated (large, medium and/or small EVs) samples into SEC separately - post isolation.
The article below may help:
Article Comparison of small extracellular vesicles isolated from pla...
If you'd like to isolate using SEC directly; you can try TFF-Easy or TFF-MV to concentrate your sample and reduce the volume 20-30 fold. I have heard of people concentrating their samples 100-fold using this method; however, you increase the risk aggregate formation in very concentrated media. (depending on your SEC column you may not be able to reduce the volume enough?)
Here's a YouTube tutorial on using TFF:
https://www.youtube.com/watch?v=nJIVf-H1PdM
Here are a couple of links for TFF purchase:
http://exotest.eu/online_orders/HBM-TFF-1
http://exotest.eu/online_orders/tff-for-ev-concentration/tff-mv
Enjoy :)
SEC is a non-concentrating technique, and the sample volume should be less than 1% of the column volume. So for 0.2 L of sample, you'd need a 20 L column. At 1300 US$ per L for the sephacryl alone not a realistic proposition.
I agree with Mier Kestecher, centrifugation followed by SEC is your best bet. Perhaps you can reduce the sample volume somewhat with an Amicon cell?
Hi,
Maybe you can first do a quick desalting step (SEC) separating "large" and "small" molecules. Using WorkBeads Dsalt (agarose-based chromatography resin) you can load 25-30% of the column volume, you will need a 0.65 - 0.7 L column if you want to load everything on the same time.
The exclusion limit of the resin is Mr 5000 for proteins and 10 bp for nucleic acids. Substances that are larger than Mr 5 000 do not enter the porous beads
and are therefore eluted in the void volume (early elution). Substances smaller than Mr 5 000 (e.g., salts, buffer substances and other low-molecular
weight additives or impurities) enter the bead pores. These substances are delayed (late elution).
Just want to add, this resin is not at all as expensive as e.g. Sephacryl resins mentioned above.
Best regards, Anna
Hi again,
Forgot to mention that using SEC in "desalting mode" make you load much larger sample volume (that I mention above), but you can also run much faster and instead of packing a long column (that can be tricky) for SEC, you use a shorter column for desalting.
Best regards, Anna
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