I used a different protocol for sample preparation of tissues of mouse origin,. Using the supplied protocol resulted in formation of insoluble material in several dilutions
1) 30 mg of frozen tissue was utilized
2) tissue was once washed in 500 µl PBS followed by centrifugation at 13.000 g for 5 min at 4°C
3) tissue was homogenized in 250 µl PBS containing 10mM NEM (N-Ethylmaleimide) and homogenized with a micro tissue lyzer
4) centrifugation at 13000 g at 4°C for 20 min and the supernatant was removed
5) 250 µl PBS containing 0,5% HTA-Br (w/v) was added
6) Samples were sonicated for 30 sec on ice
7) centrifugation for 20 min at 4°C and 8000g
8) the supernatant was utilized in a dilution of 1:2 to 1:10 for the assay directly
1) 30 mg of frozen tissue was utilized
2) tissue was once washed in 500 µl PBS followed by centrifugation at 13.000 g for 5 min at 4°C
3) tissue was homogenized in 250 µl PBS containing 10mM NEM (N-Ethylmaleimide) and homogenized with a micro tissue lyzer
4) centrifugation at 13000 g at 4°C for 20 min and the supernatant was removed
5) 250 µl PBS containing 0,5% HTA-Br (w/v) was added
6) Samples were sonicated for 30 sec on ice
7) centrifugation for 20 min at 4°C and 8000g
8) the supernatant was utilized in a dilution of 1:2 to 1:10 for the assay directly
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
FRAU DR. Katharina Rump
Verified customer
投稿 Mar 01 2016