Re: testing antibody dilution
I think your 1:5000 was over stained. The staining you saw in the
negative and in the cytoplasm was probably endogenous biotin. Since the
intensity was good though and it faded in the 1:7000 I think the first is
better but you should try to eliminate the background staining by either a
biotin block or a non biotin secondary detection system (Envision or Mach II
or an alkaline phosphotase kit).
----- Original Message -----
From: "Thu-Thao Pham"
Sent: Thursday, November 29, 2001 1:52 PM
Subject: testing antibody dilution
> Dear Histonet,
> I'm trying to figure out the right dilution for my antibody stained in
> liver cell-line. I ran several dilution and along with a positive control.
> I'm expecting my antibody to be located in nucleus. Positive control
> it's so and it also stained in cytoplasmid. On my human liver cell-line it
> showed no nucleus stain (good for me). However, there is very fainted
> stained on the cytoplasmid. Two dilution of primary antibody I don't know
> what I can conclude as my good dilution for my antibody when compare with
> the positive control.
> 1) I antibody at 1:5000 showed on a + control showed a complete staining
> nucleus and in cytoplasmid. However, negative human liver cell-line showed
> no nucleus staining (good for us), but very fainted here and there in the
> 2) I antibody at 1:7000 showed on a + control showed a complete staining
> nucleus (but not strong) and in cytoplasmid. However, negative human liver
> cell-line showed a no nucleus staining (good for us), as well as in the
> My question is, what would be my recommendation dilution for my antibody
> that I have tested after compared with my positive control. Should I go
> the 1:5000?
> Any opinion or advice would be more appreciated.
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