4 replies to this topic

[goldfinger]

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

[fishdoc]

goldfinger, on Feb 19 2010, 10:46 AM, said:

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.

[goldfinger]

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

fishdoc, on Feb 19 2010, 11:53 AM, said:

goldfinger, on Feb 19 2010, 10:46 AM, said:

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.

[fishdoc]

goldfinger, on Feb 19 2010, 11:45 AM, said:

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.

[scolix]

fishdoc, on Feb 19 2010, 07:33 PM, said:

goldfinger, on Feb 19 2010, 11:45 AM, said:

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.

yes, its right