Exosome Protein Detection Kit (CD81)

1. Experimental Preparation

Before the experiment, thaw the Exosome Protein Lysis Buffer at 4°C. Once thawed, take an appropriate amount and keep it on ice for use.

Note: It is recommended to aliquot the lysis buffer after the first thaw to avoid repeated freeze-thaw cycles.

2. Exosome Lysis

Mix the exosome sample with the lysis buffer at a 1:1 volume ratio, and incubate on ice for 10 min.

3. Protein Collection

Centrifuge at 4°C, 12,000 g (12,400 rpm) for 5 min, and collect the supernatant. The protein concentration in the supernatant can be quantified using the BCA method, and then used for Western Blot and other subsequent experiments.

Note: The above centrifugation is based on a small centrifuge (effective radius ~ 7 cm) and ≤ 2 mL centrifuge tubes. For larger volumes, it is recommended to centrifuge in aliquots.

4. Western Blot

1) Load 5 - 20 μL of CD81 positive control in parallel with experimental samples.

Note: a. Boil the CD81 positive control at 70°C for 5 min before loading.

b. When using the CD81 positive control for the first time, perform a preliminary experiment to determine the optimal loading amount for best results.

2) Recommended dilution ratio for Goat Anti-Rabbit IgG Antibody (HRP): 1:2000 – 1:5000.

3) Perform chemiluminescent detection using the ECL method (MCE Cat. No.: HY-K1005).

Note: DAB detection is not recommended due to its lower sensitivity.