Protein will stripped by overwahsing? - (Oct/31/2007 )

Some time I let transferred membrane staying in milk and TBST for hours. I am wondering if this way will strip the protein which has been transferred on the membrane?

Thank you.

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Not likely, usually block in milk and TBST o/n at 4C with no problems...

HTH

Becca

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Sometimes we have blocked for a few hours. i dont think it will strip the membrane.

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if it will be a tidious process, I think of washing/incubation for a week or more

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If it would be so easy to strip, I would no more need to use this awfull beta2 marcaptoethanol to strip !
Don't worry, you can incubate over-weekend in blocking buffer.

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You do not necessarily need bME.. 100 mM Glycin pH 2.5 for 30 min at 30 °C works fine

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Actually, long blocking times in milk CAN cause loss of antigen from the membrane - particularly if you are using a pretty high concentration of milk. It seems to be one of those quirky things about Westerns that few people are aware of. But if you've got plenty of antigen you're not likely to notice a problem. There's an old study that looked at this phenomenon specifically:

J Immunol Methods. 1989 Aug 15;122(1):129-35
http://www.ncbi.nlm.nih.gov/sites/entrez?D...Pubmed_RVDocSum

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Thank you very much for your reply.
What concentration is "pretty high"? Usually I use 5%milk for blocking. Is that high?

Thanks

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Yeah, I definitely consider 5% milk to be high. Have you tried 1%? In my experience, 1% works just as well and runs a much lower risk of antigen loss. That 1989 paper recommends 0.5% but I've never actually gone that low myself.

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Thank you Aimster for the information.

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