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incorrect blocking in immunocyto....


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#1 scistudent

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Posted 06 May 2010 - 08:53 AM

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!!

#2 mdfenko

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Posted 06 May 2010 - 10:21 AM

if your secondary antibody is made in goat then goat serum is a good blocking agent.
talent does what it can
genius does what it must
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#3 scistudent

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Posted 07 May 2010 - 01:30 AM

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....

#4 mdfenko

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Posted 07 May 2010 - 07:06 AM

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.
talent does what it can
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i do what i get paid to do

#5 Prep!

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Posted 10 May 2010 - 01:21 AM

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!!
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#6 anokhi

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Posted 16 May 2010 - 09:27 AM

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

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Posted 18 May 2010 - 10:29 AM

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.
talent does what it can
genius does what it must
i do what i get paid to do

#8 scistudent

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Posted 19 May 2010 - 02:35 AM

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!

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.






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