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incorrect blocking in immunocyto.... - (May/06/2010 )

Hi,

Had a little hiccup with my immuno :D by mistake I blocked my cells with goat serum and one of my antibodies is raised in goat.... so just wondering does anyone know can cells be re-blocked or can the goat serum be reomved? or is there no hope-should I chuck the cells? They take ages to culture so even though it's only one prep it's a hassel...
Anyone with any ideas would be great,
thanks!!

-scistudent-

if your secondary antibody is made in goat then goat serum is a good blocking agent.

-mdfenko-

Hi,

No it's my primary that is raised in goat, so my secondary is anti goat, so I'm trying to see if I can re-block with horse or something to still be able to probe for my primary without all the non specific binding to the goat serum....

-scistudent-

then, unless you have another primary you probably won't be able to use the cells. they are blocked. i know of no way to remove the block without damaging the cells.

-mdfenko-

mdfenko on May 7 2010, 09:36 PM said:

then, unless you have another primary you probably won't be able to use the cells. they are blocked. i know of no way to remove the block without damaging the cells.



i second that.. i have never come across such a thing... although if u happen to stumble upon sumthing useful.. please do share it with us!!!
Best luck!!

-Prep!-

antibody is made in goat then goat serum is a good blocking agent.
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-anokhi-

anokhi on May 16 2010, 01:27 PM said:

antibody is made in goat then goat serum is a good blocking agent.

not if it is the primary antibody that is made in goat.

-mdfenko-

Well, I tried many washes with Triton X and re-blocking... but nope , the cells were no good! so chucked them! Wont be making that mistake again!

mdfenko on May 18 2010, 10:29 AM said:

anokhi on May 16 2010, 01:27 PM said:

antibody is made in goat then goat serum is a good blocking agent.

not if it is the primary antibody that is made in goat.

-scistudent-