Western Blot Blocking - Blocking for 1hr vs overnight (Jul/30/2008 )

Hi guys,
What is the difference between blocking western blot 1hr at room temperature vs overnight at 4oC? Which gives better blocking?
Also, do you use Tween in the buffer when you block?
Thanks

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I always block overnight at 4degree. I honestly don't know if there is a huge difference between the one hour and overnight but the way I see it, you really can't over-block a membrane but you sure can under-block. Besides, it takes pretty much all day to run and transfer the gel as I always run mine nice and slow. I just put it in blocking overnight and start the probing the next morning. I use TBST for everything, including the milk buffer for blocking.

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I'm having a bit of trouble with background. I switched from milk to BSA, and that helped some, but I would like to further reduce background if possible.

I use TBST as well; I ask just because I found a page http://www.researchd.com/rdiabs/wbproto.htm that said to block without Tween, but yeah, I've always used TBST

So I'm wondering if switching from 1hr to ON would help the background. I've been in labs that did both 1hr as well as ON. The current lab I'm in is very particular about following the protocol exactly, so I'm kind of afraid to change the protocol in case it doesn't work. And I guess they probably do know more than me, since I'm just a grad student... Although I did have a feeling that ON was better, so I posted to ask what everyone else did.

Thanks.

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I completely agree with rkay. I always seem to get decent results with one hour blocking but really just feel like if you block overnight you can eliminate the blocking step as a possible source of error. As for background I have also found this helps very much so with reducing overall noise and junk. Washes are critical as well so dont be afraid to do more washes for longer peroids of time. I do three washes for ten minutes, others do five washes for 5 minutes, whatever works for you. But I would def go for overnight blocking just to be certain.

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I also do overnight blocking.

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I've found that the length of the block pretty much actually becomes negligible after about 30 mins, at room temp on a shaker. I've done overnight blocking at 4C and the issue became more washing, which isn't really much of a problem. We use a regular milk block with TTBS. What seems to be a hugh problem is making sure that before transferring the proteins to the membrane make sure the membrane has been soaked in your transfer buffer, a half hour is actually nice just to adequately "calibrate" the membrane. Other than BSA, milk, and commercial blocks, does anyone else have any good blocking strategies, anything would be nice.

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Collagen and casein are both suitable alternatives. Basically anything with non-specific protein in it will be fine.

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blocking at 4oC overnight prevents the milk from turning sour due to microbial growth. i block my membrane at room temperature for 1 hour, and i get the same results when i do it overnight at 4oC. no difference really.

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For background problems I recommend the overnight in milk blocking but you can make a big difference in your washing. Wash blots for extended periods of time with multiple buffer changes. I've washed a blot for days and saw what started out as black and uninterpretable turn to publication quality bands. You can increase the Tween a bit or even up the salt a touch in the TBS. Most background comes from the secondary so you can also decrease background by doing a second block between the primary and secondary antibodies. The second block should be the serum of the animal that the secondary was generated in (example: I use a goat anti-mouse secondary so I do a second block of 5% goat serum).

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