Amplex Red protocol

7 posts / 0 new
Last post
Guy Sovak
Guy Sovak's picture
Amplex Red protocol

Hi,
I am doing Amplex Red assay for cell surface density of Proteins.
I got some problems with high background levels. I tryed blocking before the primary Ab incubation, I increased the washing times. Nothing helps.
Looking for some Ideas.
Thanks

Guy Sovak
Guy Sovak's picture
Hi,

Hi,
First of all do at least 5 washes with Ice-Cold PBS with 1mMCa and 0.1mM Mg.
Another idea is to change your Secondary Ab to an Fab IgG.
Then maybe you can try incubating even the Substrate itself on Ice .
Let me know if it helped.

Sandy
Sandy's picture
gsovak wrote:Hi,

gsovak wrote:

Hi,
I am doing Amplex Red assay for cell surface density of Proteins.
I got some problems with high background levels. I tryed blocking before the primary Ab incubation, I increased the washing times. Nothing helps.
Looking for some Ideas.
Thanks

If your kit is from Invitrogen you can check with them and find out the best blocking protocol.

http://probes.invitrogen.com/handbook/sections/1005.html

Guy Sovak
Guy Sovak's picture
Thanks for the web page.

Thanks for the web page.
I checked it, and they are not speaking about any blocking that I didn't use yet.
Do you have any experience with amplex red and HRP?
Guy

Guy Sovak
Guy Sovak's picture
Hi,

Hi,
Just wanted to let you all know that I am slowly getting lower background.
One of the tricks is to seed the cells 2 days before the experiment in a lower dilution.
I found out the cells were not homogenous in the confluences in the well when I seeded them just one day before the experiment.
Now it is much better
Guy

luisruben14@hot...
luisruben14@hotmail.com's picture
 I have done quantitative AR

 I have done quantitative AR assays and am getting really high background with PBD, any suggestions for this?

Guy Sovak
Guy Sovak's picture
PBD ?

PBD ?
Usually there are many ways to deel with background.
I would start with washing (more times), then you can shorten the time of secoondary ab incubation.
tell me how may times you wash between each ab .
Guy