31
1. Add 50 μL of freshly prepared protein standards (10
8
–
1.35 × 10
10
exosome.mL
−1
) and exosome samples to the appropriate well of the microtiter plate.
2. Incubate the plate at 37 °C for 2 h.
3. Wash the plate three times for 5 min each with 100 μL wash
buffer.
4. Add 50 μL of 1:100 diluted CD-63 primary antibody to each
well and incubate while shaking at room temperature for 1 h.
5. Wash the plate three times for 5 min each with 100 μL wash
buffer.
6. Add 50 μL of 1:5000 diluted secondary antibody to each well
and incubate with shaking at room temperature for 1 h.
7. Add 50 μL of supersensitive TMB ELISA substrate and incubate with shaking at room temperature for 15–45 min.
8. Add 50 μL of stop buffer and read the absorbance values at
450 nm immediately (Lambda Scan Instrument).
1. Add 2× Laemmli buffer to the exosome suspensions (18 μg),
and then boil the samples at 96 °C for 3 min.
2. Centrifuge the exosomal protein solutions at 13,000 × g for
3 min; take the supernatant into clean microtubes.
3. Load 25 μL of each sample into each well (5–12% gel system is
used) and then run proteins at 100 V, 35–40 mA, for about 2 h
(Bio-Rad Wet/Tank Blotting System).
4. Transfer the proteins electrophoretically (100 V, 400 mA) for
1 h onto immobilon PVDF membrane.
5. Block the membrane with 5% nonfat milk powder in TBS-T for
1 h on a platform shaker.
6. Incubate the membrane with CD63 and CD81 antibodies
overnight at 4 °C.
7. Following the washing step with TBS-T, incubate the membrane with secondary antibody diluted in PBS for 1 h at 4 °C.
8. After washing with PBS, the membrane is treated with ECL
substrate and imaged on an Odyssey imaging system.
1. The exosome suspension is treated with 4% paraformaldehyde
for fixation.
2. Deposit 10 μL of the fixed exosomes onto Formvar carboncoated grids and wait for 20 min for adequate absorption.
3. Put 100 μL of PBS onto a piece of parafilm and place the grid
on PBS drop with a clean forceps. Repeat the washing step
three times.
3.4 Western Blot
Analysis
3.5 Preparation
of TEM Samples
Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole…
1. Add 50 μL of freshly prepared protein standards (10
8
–
1.35 × 10
10
exosome.mL
−1
) and exosome samples to the appropriate well of the microtiter plate.
2. Incubate the plate at 37 °C for 2 h.
3. Wash the plate three times for 5 min each with 100 μL wash
buffer.
4. Add 50 μL of 1:100 diluted CD-63 primary antibody to each
well and incubate while shaking at room temperature for 1 h.
5. Wash the plate three times for 5 min each with 100 μL wash
buffer.
6. Add 50 μL of 1:5000 diluted secondary antibody to each well
and incubate with shaking at room temperature for 1 h.
7. Add 50 μL of supersensitive TMB ELISA substrate and incubate with shaking at room temperature for 15–45 min.
8. Add 50 μL of stop buffer and read the absorbance values at
450 nm immediately (Lambda Scan Instrument).
1. Add 2× Laemmli buffer to the exosome suspensions (18 μg),
and then boil the samples at 96 °C for 3 min.
2. Centrifuge the exosomal protein solutions at 13,000 × g for
3 min; take the supernatant into clean microtubes.
3. Load 25 μL of each sample into each well (5–12% gel system is
used) and then run proteins at 100 V, 35–40 mA, for about 2 h
(Bio-Rad Wet/Tank Blotting System).
4. Transfer the proteins electrophoretically (100 V, 400 mA) for
1 h onto immobilon PVDF membrane.
5. Block the membrane with 5% nonfat milk powder in TBS-T for
1 h on a platform shaker.
6. Incubate the membrane with CD63 and CD81 antibodies
overnight at 4 °C.
7. Following the washing step with TBS-T, incubate the membrane with secondary antibody diluted in PBS for 1 h at 4 °C.
8. After washing with PBS, the membrane is treated with ECL
substrate and imaged on an Odyssey imaging system.
1. The exosome suspension is treated with 4% paraformaldehyde
for fixation.
2. Deposit 10 μL of the fixed exosomes onto Formvar carboncoated grids and wait for 20 min for adequate absorption.
3. Put 100 μL of PBS onto a piece of parafilm and place the grid
on PBS drop with a clean forceps. Repeat the washing step
three times.
3.4 Western Blot
Analysis
3.5 Preparation
of TEM Samples
Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole…