[Histonet] New rabbit antibody... but what do I use as negative (isotype) control?

[Andrea T. Hooper]

Hi Gayle,

I couldn't agree more with what you have said below ... but what is 
the reason for heat inactivation? There is no worry of complement in 
dead, sectioned, fixed tissues. I don't HI my serum and have no 
problems with background.

Thanks,
Andrea



At 9:41 AM -0600 5/11/05, Gayle Callis wrote:
>Rabbit IgG will work - you can purchase from Jackson.  This is a 
>polyclonal, and if there is no isotype listed, whole IgG will 
>suffice.
>
>The concentration of the IgG should match the concentration of your 
>primary antibody, and calculate it in terms of ug/ml - a little 
>math. Rather than rely on some dilution, you will dilute the Rb IgG, 
>but its original concentration in mg/ml may be much higher than the 
>primary antibody's mg/ml, meaning you might be using a different 
>dilution for Rb IgG than you use for the rabbit primary antibody.
>
>You could use rabbit serum, but it should be heat inactivated - if 
>the polyclonal is affinity purified and the pooled serum is not, you 
>may get more nonspecific binding with serum.  Rabbit IgG would be my 
>choice.
>

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