The colour of Caspase-9 assay - (Mar/22/2007 )
When i use colorimetric assay to detect the activation of caspase-9(My substrate for caspase-9 is Ac - LEHD - pNA), I have a problem that i can not make it clear
We can not see the yellow colour like the result of caspase-3 assay. The result of caspase-9 is a muddy colour.
Can u help me solve this problem?
OD405 nm for product measurement. The turbidity maybe due to your sample is not completely in solution. Have you spinned it before the reaction? Take supernatant only.
I have followed your instruction. I use bradford assay to determine the amount of protein. I have 2 samples, the first sample, i use 100 micro gram protein, the second sample i use 300 micro gram protein. After adding the coloric subtracte of caspase-9, the first sample is turbidity; the second sample is dime yellow (OD 405 of the second sample more two fold than the control)
I cannot understand why the first sample which has less protein than the first sample is turbidity. In the other hand, the second sample which has more protein is dime yellow.
Could you help me to overcome this problem.
can you tell us the source of the samples and how did you prepare them?
Hela cells which have 70-80% confluence are treated witn camptothecin with IC 50 for 12, 24,36, 48 hours. After that, i collect the cells and rinse with PBS, centrifugate and remove PBS, add buffer lysis and keep the sample in ice for 3 minutes,then keep in warm water (370C) (we perform 4 times). The samples are centrifuged 4oC, 10 minutes 15000g, We collect the supernant. The amount of protein is determined by Brad ford assay. at last, We add 50 micro lit sample with 5 microlit caspase 9 substrate Ac-LEHD-pNa