IL 8 - western blotting - (Dec/09/2013 )

Dear Friends,

this is nagu, asking some help regarding IL8 targetting in Western bloting

Every one is using ELISA for IL8.

But we had a antibodies, so i decided to do western bloting.

As I am using AGS cell line, it expresses IL8 and can be detected in ELISA.

But in western blotting i cant see the postive signal in both cell lysate and concentrated supernatant.

conditions

blocking 1 hour

Primary - 1:2000, over night

Secondary - 1: 3000 - 1 hour.

Washing 10 min - 3 times

can any one help me to out of my problem.

thanks in advance.

nagu

in general, elisa detects native protein, western blot detects denatured protein. does the data sheet for your antibody say that it can be used for both? if not then you should purchase an antibody which has been qualified for western blot.