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Incubation with washing buffer only?


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#1 goldfinger

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Posted 19 February 2010 - 08:46 AM

Hi all,
I am using the sigma's anti flag antibody (F7425) which is produced in rabbit. The data sheet shtates that for Anti-Rabbit HRP binding, the incubation is done in PBS w/ 0.05%TWEEN20.
However, I always done incubation in washing buffer with 5% mlik or BSA.

I am wondering why BSA or milk is not required here or why they are required in other protocols.
Thanks

#2 fishdoc

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Posted 19 February 2010 - 08:53 AM

Hi all,
I am using the sigma's anti flag antibody (F7425) which is produced in rabbit. The data sheet shtates that for Anti-Rabbit HRP binding, the incubation is done in PBS w/ 0.05%TWEEN20.
However, I always done incubation in washing buffer with 5% mlik or BSA.

I am wondering why BSA or milk is not required here or why they are required in other protocols.
Thanks



You can do it either way. Using blocking buffer will result in less background, but also may reduce signal a bit. Most of the instructions given with the antibodies are general to apply across most applications. In most instances, optimization is required for best results, and this is one of those things that can be tweaked for better results.

#3 goldfinger

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Posted 19 February 2010 - 09:45 AM

Thanks for answering my questions, :)
So the BSA or milk are for blocking the non-specific binding, not for protecion of the antibody. Right?


Hi all,
I am using the sigma's anti flag antibody (F7425) which is produced in rabbit. The data sheet shtates that for Anti-Rabbit HRP binding, the incubation is done in PBS w/ 0.05%TWEEN20.
However, I always done incubation in washing buffer with 5% mlik or BSA.

I am wondering why BSA or milk is not required here or why they are required in other protocols.
Thanks



You can do it either way. Using blocking buffer will result in less background, but also may reduce signal a bit. Most of the instructions given with the antibodies are general to apply across most applications. In most instances, optimization is required for best results, and this is one of those things that can be tweaked for better results.



#4 fishdoc

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Posted 19 February 2010 - 10:33 AM

Thanks for answering my questions, :)
So the BSA or milk are for blocking the non-specific binding, not for protecion of the antibody. Right?



I'm relatively new to westerns, but yes, that is my understanding.

Edited by fishdoc, 19 February 2010 - 10:34 AM.


#5 scolix

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Posted 19 February 2010 - 11:57 AM

Thanks for answering my questions, :D
So the BSA or milk are for blocking the non-specific binding, not for protecion of the antibody. Right?



I'm relatively new to westerns, but yes, that is my understanding.


yes, its right




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