Anti CD81, Rat Monoclonal Antibody (9B) Application Data

Western Blot

Isolated exosomes derived from culture supernatant, human serum, and human heparin plasma were electrophoresed under non-reducing conditions. And Western Blot was performed using this antibody.

Sample	Exosomes isolated from cell culture supernatant of COLO201 cells, human serum, and human heparin plasma
Primary antibody	Anti CD81, Rat Monoclonal Antibody (9B) (Catalog ID 010-28223) (1 μg/mL)
Secondary antibody	Anti Rat IgG (H+L), Donkey, Peroxidase-conjugated [code No. 564-73961] (1:20,000)
Detection reagent	ImmunoStar Zeta [code No. 291-72401]

CD81 protein (26 kDa) in each samples (non-reducing condition) was detected with this product.

Data of cross-reactivity

The cross-reactivity of this product with human, mouse, rat, and bovine serum was detected.

Sample	Human serum, Mouse serum, Rat serum, and Bovine serum
ELISA Kit	PS Capture Exosome ELISA Kit (Streptavidin HRP) (Catalog ID 298-80601)
Detection antibody	Anti CD81, Rat Monoclonal Antibody (9B) (Catalog ID 010-28223) –biotin labeled (250 ng/mL)

The signal to human serum was set to 100%.
No signals for mouse and rat serum were obtained, and bovine serum responded slightly.
Although this antibody responds very slightly to bovine serum, it has been shown to be highly specific for human serum.

Reactivity to FBS in culture medium

The reactivity of the antibody with FBS in the culture medium was confirmed.

Sample	A: The culture medium containing 15% FBS B: Culture supernatant of cells cultured in a culture medium containing 15% FBS After A and B were concentrated 10 times, then the exosomes were isolated with our kit [MagCapture Exosome Isolation Kit PS [code No. 293-77601]]. The obtained recovered samples were electrophoresed under non-reducing conditions.
Primary antibody	Competitor: Anti-CD81 antibody (competitor) (1 μg/mL) This product: Anti CD81, Rat Monoclonal Antibody (9B) (Catalog ID 010-28223) (1 μg/mL)
Secondary antibody	HRP-conjugated Anti Mouse / Rat IgG antibody
Detection reagent	ImmunoStar Zeta [code No. 291-72401]

The competitor’s antibody, which does not guranteed specificity to cross-reactivity was detected the exosomes from both A and B.
This product, which has been confirmed to have low cross-reactivity with bovine CD81 did not detect the exosomes from A. And it detected the exosomes from B.

*The reason for increasing the exposure time is to confirm that this antibody does not detect bovine CD81 from FBS.

It was shown that the background caused by FBS added to the culture medium was not detected by using this antibody.