Wako Pure Chemical Industries, Ltd.Wako Pure Chemical Industries, Ltd. - Laboratory Chemicals
Home  > Products >  Life Science > Genetic Research  >  MagCapture™ Exosome Isolation Kit PS

Exosome isolation by novel affinity molecule
MagCapture™
Exosome Isolation Kit PS

Exosomes and other extracellular vesicles (EVs) are small membrane vesicles containing protein, mRNA, micro RNA, DNA and lipids, which are secreted by various cells and are stable in body fluids including blood, saliva, urine, cerebrospinal fluid (CSF) and breast milk. These EVs have been recognized as messengers of cell-to-cell communication, and biomarkers for various diseases. Conventionally, ultracentrifugation, affinity purification using antibody to surface antigen, and precipitation with polymer reagents are used for isolation of exosomes. However, these methods are not fully satisficing in all performances criteria such as recovery efficiency, purity and operability.
MagCapture™ Exosome Isolation Kit PS adopts a novel affinity purification method using magnetic beads and phosphatidylserine (PS)-binding protein (PS affinity method). This kit can easily isolate high purity exosomes and other EVs from cell culture medium and body fluids at high yield by a normal microcentrifuge. If higher purity exosomes are needed, please use the 10,000 x g supernatant as sample. This kit enables the isolation of exosomes and other EVs as intact forms because the captured EVs are eluted from magnetic beads with metal-chelating reagent at neutral pH. The isolated intact exosomes and other EVs can be used for various applications including electron microscopic analysis, nanoparticle tracking analysis, administration of EVs and analysis of molecular constituents such as proteins, lipids, or nucleic acids.


1. Affinity method for phosphatidylserine (PS) on membrane surface

Affinity method

Using Phosphatidylserine (PS)-binding protein, extracellular vesicles are captured in a metal ion-dependent manner, followed by eluting them with metal ion chelating reagent.

2. Features

Novel affinity method

· Recovery by a PS-binding molecule
· Low background
· Mild elution by a chelating reagent on a neutral pH condition


High purity of intact exosomes

Not required ultracentrifugation

· Improved operation by using magnetic beads
· Optimized protocol

High reproducibility


Comparison with other purification method
Methods Exosome purity Exosome
recovery
Intact vesicles recovery
MagCapture™ (PS affinity method) ■■■■■ ■■■■
Yes
Ultracentrifugation ■■ ■■
Yes
Polymer-based precipitation ■■■■■
Yes
Exosome surface antigen affinity method
(using the antibody)
■■■
No

Target samples: cell culture supernatant, serum, urine, etc.
NOTE: This kit is NOT suitable for exosome isolation from plasma treated with chelating agents such as EDTA and citric acid.

3. Product List

Product Name Package Size Wako Cat. No. Storage
MagCapture™ Exosome Isolation Kit PS
10 tests
Keep at 2-10°C.
  [Kit Contents]
    (1) Streptavidin Magnetic Beads
    (2) Biotin-labeled Exosome Capture
    (3) Exosome Capture Immobilizing Buffer
    (4) Exosome Binding Enhancer (x 500)
    (5) Washing Buffer
    (6) Exosome Elution Buffer
    (7) Reaction Tubes
< 10 purifications >
600 μL x 1 tube
100 μL x 1 tube
35 mL x 1 bottle
500 μL x 1 tube
75 mL x 2 bottles
5 mL x 1 bottle
22 tubes
 

4. The yield comparison of exosome isolated from human serum

Exosomes were isolated from human serum by using MagCapture™, ultracentrifugation and antibody affinity method, followed by western blot with the anti CD9, anti CD63 and anti CD81 antibodies.
CD9, CD63 and CD81 are exosome markers.
Lane 1: Ultracentrifugation
Lane 2: MagCapture™
Lane 3: Exosome Isolation kit (CD9) [Company A]
Lane 4: Exosome Isolation Kit (CD63)[Company A]
Lane 5: Exosome Isolation Kit (CD81)[Company A]
Lane 6: Exosome Isolation Kit (Antibody beads-mixture of CD9, CD63, CD81 & EpCAM)

5. The performance comparison with conventional exosome isolation methods

The yield and purity were compared for exosomes isolated from K562 (human chronic mylogenous leukemia: CML) cell culture supernatants (serum-free medium, or 10% Exosome-depleted FBS medium) by using MagCapture™, ultracentrifugation and polymer-based precipitation method..


MagCapture™ Exosome Isolation Kit PS
Exosomes were collected from 1 mL of pretreated (10,000 x g, 30 min.) K562 cell culture supernatant (serum-free medium or 10% exosome-depleted FBS medium) by using MagCapture™ standard protocol (reaction time: 3 hours)


Ultracentrifugation
Exosome fractions were collected from 10 mL of pretreated (10,000 x g, 30 min.) K562 cell culture supernatant (serum-free medium or 10% exosome-depleted FBS medium) by ultracentrifugation (110,000 x g, 70 min.). The precipitates were suspended by TBS and then exosomes were recovered by ultracentrifugation (110,000 x g, 70 min.) asa pellet.


Polymer-based precipitation
Exosomes were collected from 1 mL of pretreated (10,000 x g, 30 min.) K562 cell culture supernatant (serum-free medium or 10% exosome-depleted FBS medium) by using Company A’s product protocol.(precipitation time: overnight).


5-1. Electron microscopic analysis and Nano analysis of isolated exosomes


The particle size of exosomes from K562 cell culture supernatant (serum-free medium) using MagCapture™, ultracentrifugation and polymer-based precipitation, respectively was determined by using NanoSight LM-10. The collected exosomes (2-4 x 1010 particles) were fixed by 2% paraformaldehyde and analyzed by electron microscopy.

5-2. The comparison of recovery amounts and purity of exosomes (1)


Exosomes were collected from K562 cell culture supernatant (serum-free medium) by MagCapture™, ultracentrifugation and polymer-based precipitation. The recovery efficiency and purity were analyzed by silver staining and western blotting by using anti CD63, anti Flotillin-2 and anti Lamp-1 antibodies).

5-3. The comparison of recovery amounts and purity of exosomes (2)


Exosomes were collected from K562 cell culture supernatant (10% exosome-depleted FBS medium) by MagCapture™, ultracentrifugation and polymer-based precipitation . The recovery efficiency and purity of exosomes were analyzed by silver staining and western blotting by using anti CD63, anti Lamp-1 and anti Flotillin-2 antibodies. Furthermore, collected sample from each was analyzed by mass spectrometry and compared the percentage of human-derived peptides derived from K562 cells.

6. Pretreatment protocol from cell culture supernatant or serum

7. Pretreatment protocol for Plasma by buffer exchange with TBS buffer

(79KB/1p)

Step 1:   Add 19 mL of TBS buffer to VIVASPIN 20 (100K).
Step 2:   Add 1 mL of plasma to VIVASPIN 20 (100K) from step 1 and mix.
Step 3:   Centrifuge VIVASPIN 20 (100K) from step 2. (Refer to a centrifugal condition in the manufacturer's instruction manual.)
Step 4:   Add 10 mL of TBS buffer to VIVASPIN 20 (100K) from step 3 when the upper liquid dropped.
Step 5:   Centrifuge.
Step 6:   Add 10 mL of TBS buffer to VIVASPIN 20 (100K) from step 5 when the upper liquid dropped.
Step 7:   Centrifuge.
Step 8:   Add 11 mL of TBS buffer to VIVASPIN 20 (100K) from step 7 when theupper liquid dropped.
Step 9:   Centrifuge VIVASPIN 20 (100K) until the volume of sample become under 1 mL.

      ⇒ 50 mL of TBS buffer in total is used in this protocol.

8. Exosome Isolation Flow Chart

flow

9. Reference

W. Nakai, T. Yoshida, D. Diez, Y. Miyatake, T. Nishibu, N. Imawaka, K. Naruse, Y. Sadamura & R. Hanayama, Sci Rep, 6, 33935 (2016).

10. Product List

Product Name Package Size Wako Cat. No. Storage
MagCapture™ Exosome Isolation Kit PS
10 tests
Keep at 2-10°C.
  [Kit Contents]
    (1) Streptavidin Magnetic Beads
    (2) Biotin-labeled Exosome Capture
    (3) Exosome Capture Immobilizing Buffer
    (4) Exosome Binding Enhancer (x 500)
    (5) Washing Buffer
    (6) Exosome Elution Buffer
    (7) Reaction Tubes
< 10 purifications >
600 μL x 1 tube
100 μL x 1 tube
35 mL x 1 bottle
500 μL x 1 tube
75 mL x 2 bottles
5 mL x 1 bottle
22 tubes
 

11. Related Products

Tim-Fc series

These products are fusion proteins which were consisted of the extracellular domain of human or mouse TIM family (TIMD1, TIMD3, or TIMD4) and the Fc region of human IgG1 or mouse IgG2a. The Fc region contains a hinge sequence. The TIM (T cell/transmembrane, immunoglobulin and mucin) family plays a critical role in regulating immune responses, including allergy, asthma, transplant tolerance, autoimmunity and the response to viral infections. The unique structure of TIM immunoglobulin variable region domains allows highly specific recognition of phosphatidylserine (PtdSer), exposed on the surface of apoptotic cells.

Product Name Pkg. Size Wako Cat. No. Grade Storage
Human TIM1/Human Fc Chimera, recombinant 100 μg 080-10231 for Genetic Research Keep at -20°C.
Human TIM3/Human Fc Chimera, recombinant 100 μg 087-10241
Human TIM3/Mouse Fc Chimera, recombinant 100 μg 084-10251
Human TIM4/Human Fc Chimera, recombinant 100 μg 081-10261
Mouse TIM1/Human Fc Chimera, recombinant 100 μg 137-18491
Mouse TIM3/Human Fc Chimera, recombinant 100 μg 130-18501
Mouse TIM4/Human Fc Chimera, recombinant 100 μg 137-18511

Control Protein (human IgG1-Fc, mouse IgG2a-Fc)

These products are recombinant proteins of Fc region of human immunoglobulin G1 or mouse immunoglobulin G2a. It should be used as the control protein for the western blotting or the activity test of human Fc or mouse Fc chimeric proteins. These products are lyophilized from 0.2μm-filtered solution in PBS. The non-lytic type has mutations to the complement (C1q) and FcgR I binding sites of the IgG Fc fragment. These mutations render the proteins incapable of antibody directed cytotoxicity (ADCC) and complement directed cytotoxicity (CDC).

Product Name Pkg. Size Wako Cat. No. Grade Storage
IgG1 Fc, Human, recombinant 100 μg 098-07141 for Genetic Research Keep at -20°C.
IgG1 Fc (non-lytic), Human, recombinant 100 μg 095-07151
IgG2a Fc, Mouse, recombinant 100 μg 092-07161
IgG2a Fc (non-lytic), Mouse, recombinant 100 μg 099-07171

A novel tool for qualitative and quantitative analysis of extracellular vesicles in sample of cell culture supernatant

Product Name Pkg. Size Wako Cat. No. Grade Storage

PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD)

HTML
96 tests 297-79201 for Genetic Research Keep at
2-10°C.
Home  > Products >  Life Science > Genetic Research  >  MagCapture™ Exosome Isolation Kit PS