aboyt at uniwa.uwa.edu.au
Wed Oct 4 04:14:13 EST 1995
I am currently carrying out westerns using Pierce ECL visualisation.
I use two methods.
1) Primary Ab, wash 2x briefly TBST (0.1%) then 3 x 10 min
Secondary Ab conjugated to HRP in TBST, same washing as before.
2) Primary and wash as above
Biotinylated secondary in TBS, wash as above
Strepdavidin-HRP in TBST, wash as above
Primary Ab conc is 1/1000 (ie 1ug/mL)
Secondary is 1/5000
I expect the second method to be more sensitive (due to the Strep-Biotin
step), but it isn't. If anything it is slightly less sensitive.
My biotinylated secondary is from Dako (Rabbit anti-mouse)
My HRP conjugated secondary is fromn Amersham.
All blocking (and Primary antibody step) is in 10% milk.
If Anyone could help it would be appreciated (its driving me crazy!)
(aboyt at uniwa.uwa.edu.au)
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