dilution for western

Shaun D. Black SHAUN at JASON.UTHCT.EDU
Wed Jun 22 16:10:30 EST 1994


On 6/22/94, Matthias Pfeiffer wrote:
> 
> Recently I made an ELISA with an polyclonal antiserum. I got a quite good 
> reaction with a 3200 fold dilution. Now I want to do western blotting. Since 
> I haven't got experience I want to know which dilution of the antiserum I 
> have to use for this. I've checked an 800 fold dilution but didn't get a 
> signal. Could anyone help me? Thanks in advance.
> 
I'm a bit surprised that you got no signal at all at with the 1:800 dilution,
but it is important to note that Westerns are considerably less sensitive
than ELISA.  Thus, try less dilute polyclonal in your Westerns.  (On rare 
occasions, we have been forced to use 1:20 dilutions of certain antibody
preps for Western blots).  Also, your detecting antibody and substrate will
control to a significant degree the sensitivity level you may achieve.  In
this connection HRP conjugates are less sensitive than alkaline phosphatase
conjugates.  With HRP, ABTS is not as sensitive a substrate as DAB (and its
derivatives).  Hope this helps you!   Best regards,  Shaun
                                                     (shaun at jason.uthct.edu)



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