5. Dissolve the pellets depending on the proteomic approach: for
2D-DIGE, use 40μL of DIGE buffer; for label-free LC-MS/
MS, use 40μL of LC-MS/MS buffer.
6. Store EV lysates at À80
C.
3.3 EV
Characterization
After isolation, there are several methods to characterize the heterogeneity of the EV sample. The ISEV recommends using at least
two different technological approaches in order to characterize EV
samples [2]. The principal techniques for characterization are electron microscopy-based (transmission electron microscopy (TEM)
and cryo-TEM), particle enumeration techniques (e.g., nanoparticle tracking analysis (NTA), dynamic light scattering (DLS)), and
flow cytometry [12].
3.4 Gel-Based
Proteomics: 2D-DIGE
Labeling
The example shown is based on a 2D-DIGE proteomic protocol
where two different conditions are compared (e.g., health and
disease). In this context, six gels were run as technical replicates
using a total amount of 150μg of protein per gel. Due to the
amount of protein required, pools can be used (see Note 6). All
steps must be performed in the dark.
1. Label randomly 50μg of the protein mixture from each condition with 400 pmol minimal CyDye DIGE fluors Cy3 and Cy5.
In addition, label 50μg of the third condition, which is a pool
of both samples (internal standard) with 400 pmol Cy2. The
labeling is on ice for 30 min.
2. Stop the reaction with 1μL of 10 mM lysine on ice for 10 min.
3. After the labeling step, pool the three samples labeled and add
an equal volume of 2Â sample buffer. Leave the mix for 15 min
on ice.
4. Before reswelling of IPG strips, dilute the samples in 2D sample
buffer to 500μL (final volume), and add ampholytes to a final
concentration of 1.6% (v/v).
3.5 2D-Electrophoresis
1. Rehydrate the 24-cm 4–7 IPG strips with the sample for 16 h
(see Note 7).
2. Run the first dimension (isoelectric focusing (IEF)) powered
by the Multiphor II for 64.9 kVh at 17
C (see Note 8).
3. After the first dimension, equilibrate the strips with the reduction buffer for 15 min with gentle agitation.
4. Wash the excess with ultrapure water and, after that, equilibrate
the strips with the reduction buffer for 15 min with gentle
agitation.
5. Wash the strips again with ultrapure water.
6. Place the strips on the top of the second-dimension gels with
0.5% of melted agarose.
18
Maria N. Barrachina and A ´ ngel Garcı ´a
2D-DIGE, use 40μL of DIGE buffer; for label-free LC-MS/
MS, use 40μL of LC-MS/MS buffer.
6. Store EV lysates at À80
C.
3.3 EV
Characterization
After isolation, there are several methods to characterize the heterogeneity of the EV sample. The ISEV recommends using at least
two different technological approaches in order to characterize EV
samples [2]. The principal techniques for characterization are electron microscopy-based (transmission electron microscopy (TEM)
and cryo-TEM), particle enumeration techniques (e.g., nanoparticle tracking analysis (NTA), dynamic light scattering (DLS)), and
flow cytometry [12].
3.4 Gel-Based
Proteomics: 2D-DIGE
Labeling
The example shown is based on a 2D-DIGE proteomic protocol
where two different conditions are compared (e.g., health and
disease). In this context, six gels were run as technical replicates
using a total amount of 150μg of protein per gel. Due to the
amount of protein required, pools can be used (see Note 6). All
steps must be performed in the dark.
1. Label randomly 50μg of the protein mixture from each condition with 400 pmol minimal CyDye DIGE fluors Cy3 and Cy5.
In addition, label 50μg of the third condition, which is a pool
of both samples (internal standard) with 400 pmol Cy2. The
labeling is on ice for 30 min.
2. Stop the reaction with 1μL of 10 mM lysine on ice for 10 min.
3. After the labeling step, pool the three samples labeled and add
an equal volume of 2Â sample buffer. Leave the mix for 15 min
on ice.
4. Before reswelling of IPG strips, dilute the samples in 2D sample
buffer to 500μL (final volume), and add ampholytes to a final
concentration of 1.6% (v/v).
3.5 2D-Electrophoresis
1. Rehydrate the 24-cm 4–7 IPG strips with the sample for 16 h
(see Note 7).
2. Run the first dimension (isoelectric focusing (IEF)) powered
by the Multiphor II for 64.9 kVh at 17
C (see Note 8).
3. After the first dimension, equilibrate the strips with the reduction buffer for 15 min with gentle agitation.
4. Wash the excess with ultrapure water and, after that, equilibrate
the strips with the reduction buffer for 15 min with gentle
agitation.
5. Wash the strips again with ultrapure water.
6. Place the strips on the top of the second-dimension gels with
0.5% of melted agarose.
18
Maria N. Barrachina and A ´ ngel Garcı ´a