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Sanjay Vijendran 10-03-2005 07:04 PM

Blocking of epoxy-coated slides
 
Hi all,

I'm attempting some protein binding experiments using epoxy-coated
slides and am having trouble with blocking the slides effectively
after printing them with spots. I'm printing spots of Biotin onto the
slides and have tried blocking with both1%BSA in PBS solution as well
as ethanolamin in sodium borate buffer, but I still have lots of
non-significant binding from streptavidin. The blocking time I have
used is 30 minutes.

Has anyone had success with blocking streptavidin binding after
attaching biotin (or biotinylated proteins) to epoxy slides? I'd be
interested in hearing about any other good protocols for doing
streptavidin-biotin binding on either epoxy or aldehyde coated slides.

Thanks very much.

Sanjay


DK 10-05-2005 03:47 AM

Blocking of epoxy-coated slides
 
In article <[Only registered and activated users can see links. Click Here To Register...].net> , [Only registered and activated users can see links. Click Here To Register...] wrote:

My bet that you beckground is coming from non-covalent binding.
No experience with epoxy slides but from the Western experience
I can definitely say that 1% BSA is not sufficient. Both for PVDF and
nitrocellulose, background signal is a lot less when one uses
3% BSA or 5% dry milk. 30 min is also a bit too short. 1 hr will be
better. Surely doing everythign in ethanoleamine buffer pH ~ 8.0
won't hurt in your case.

DK





Louis Hom 10-05-2005 12:41 PM

Blocking of epoxy-coated slides
 

It's hard to tell if you've tried this already, but I would quench the
unreacted epoxides with the ethanolamine (I've used a 50 mM solution pH'd
to 8.5, no other buffer present) and then block with 1-3% BSA. Also
include the BSA in your streptavidin solution. You might also consider
turning the conjugation around and functionalize the surface with amino
groups and immobilize NHS-biotin.
--
__________________________________________________ ____________________________
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