Isolation tools

Isolation tools

What are the current exosome Isolation products available from HBM?

We offer a wide portfolio of products specifically for exosome isolation. Our Isolation tools provides solutions for isolating exosomes by multiple techniques, such as immunoaffinity, chemical precipitation, size exclusion chromatography (SEC). For more detailed information, please go to http:// http://exotest.eu/online_orders/exosome-capture-and-isolation.

Could the isolated exosome pellet be visible to naked eyes from human plasma and serum using EXO-PREP Reagent?

Yes, pellet is in general visible from a minimum amount of 100 µl of plasma and 250 µl of serum.

Could the isolated exosome pellet be visible to naked eyes cell media and urine using EXO-PREP Reagent?

Pellet is lightly visible from 1 ml of cell medium or 5 ml of urine.

How to make sure that the exosomes are isolated successfully by your exosome isolation tools?

You can perform analysis for checking the exosome size (NTA). You can do a western blot analysis on the isolated, purified exosomes using antibodies to exosomal markers (CD81, ALIX, TSG101, CD9). You can check your vesicles by electron microscopy (TEM).

Is there any specific marker for exosomes isolated from adipocytes?

It has been shown that almost all adipocyte-derived exosomes express fatty acid binding protein (FABP4).

Why specific isolation of exosomes is important?

If you are interested in selective and specific exosomal marker expressing exosomes, then we recommend immunocapture methods of exosomes isolation i.e. using immunobeads or immunoplates. However, same exosomes can express more than one exosomal markers.

Do you have exosome isolation reagents from Stem Cell Media?

Yes, we do have exosome isolation kit for isolating exosomes from stem cell media.

What are the advantages of HansaBioMed exosome isolation tools as compared to ultracentrifugation?

· Ready to use, easy and fast protocol

· Time and money saving

· We can process more than a few samples at a time

· Small starting sample volume

The data sheet says up to 100 µl of starting sample volume. Do you have any information on how much protein or RNA that yields? Also have you tested with lower or higher sample volumes?

100 µl is the best volume of fluids to load on ELISA plates. Loading 200 µl does not substantially change the vesicle yield, but increases the background. Loading 50 µl is possible, with a consequent reduction of the signal in ELISA assay. About RNA yield: it is possible to isolate RNA from the vesicles captured onto the immunoplates. But, since the vesicle quantity in each well is low, we recommend collecting the vesicles lysates from at least 4 wells to have a consistent RNA yield.

Have you attempted to elute intact exosomes from the ELISA plate?

Although HBM does not provide any tools for eluting exosomes from the ELISA plate, the elution is possible. Customer can try making it with buffers containing a small percentage (less than 0.05 %) of Tween 20. Due to the low yield of vesicles in each well, we recommend collecting together the eluted vesicles for at least 4 wells to proceed with further analyses. We cannot say for sure if this method works. However, using immunobeads, you can purify exosomes and elute from the immunobeads.

How do you isolate and quantify exosomes in one step?

You can use HBM ExoTEST kits for the quantitative and qualitative analysis of exosomes from small amount of human biological fluids (plasma, serum, urine, saliva) or cell media. Transparent and white plates are available depending on the downstream detection approach (colorimetric and luminometric respectively).

What is the minimum volume of starting sample that can be used for exosome isolation?

The data sheet says up to 100 µl of starting sample volume. The quantity of exosomes could vary between samples. Thus, we always recommend a larger starting amount of sample especially for urine and cell supernatant samples. For urine and cell media, we recommend starting with at least 1 ml of the sample volume. Concentrate the urine sample or cell supernatants about 10-20 fold using a spin concentrator. Then load 50-100 µl per well in the ELISA plate. Otherwise, for most of the body fluids the minimum volume tested is 50-100 µl per well. We recommend adding 1X PBS if the volume is less than 100 μl.

How to isolate and purify exosomes from tissue samples?

Since exosomes are secreted, we recommend performing short term tissue explant cultures to isolate exosomes from the tissues. The tissue explants in culture will secrete exosomes. You can collect the cell supernatant and isolate exosomes using the protocol similar to the exosome isolation protocol from cell media.

What are the negative control for exosome isolation?

The below mentioned proteins should be absent from the Exosome preparation http://www.journalofextracellµlarvesicles.net/index.php/jev/article/view/26913 Endoplasmic reticulum; Grp94HSP90B1; calnexin (CANX); Golgi (GM130); Mitochondria (cytochrome CCYC1); Nucleus (histonesHIST*H*); Argonaute/RISC complex (AGO*).

HansaBioMed offers two different size of immunobeads for one kind of sample, 0.4 and 1.0 micron? How to choose the size of the beads to isolate one kind of sample, for example from biofluids?

Yes, we do offer two different sizes of the Immunobeads: 0.4 and 1 micron. The choice of the immunobead size depends on your final analysis. For WB, RNA extraction and profiling, for eluting intact exosomes we recommend the usage of 0,4 um beads.  This is because if you use 0.4 micron beads, the number of beads added per volume is more and the total surface for 0.4 micron is bigger than 1.0 micron. Additionally, they capture better from small volume of samples. 1 micron beads are useful for exosome FACS analysis.