ECL Optimisation

Adam Boyt 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!)

Adam Boyt
(aboyt at uniwa.uwa.edu.au)



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