Hi, everybody! Monomeric avidin bind to biotin with moderate affinity (Kd~100 nM). Can I fuse a protein with monomeric avidin for conjugating the protein with biotinylated molecules? Will it provide stable coupling? Thanks in advance!
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#2
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an elder
Posted 29 January 2010 - 07:00 AM
you don't need to bind avidin to your protein to biotinylate it. you can biotinylate directly.
besides, avidin is relatively large (even monomeric) and may interfere with protein function.
avidin is often immobilized on a resin and is sometimes conjugated with an enzyme (eg-hrp) to use for detection of biotinylated proteins.
besides, avidin is relatively large (even monomeric) and may interfere with protein function.
avidin is often immobilized on a resin and is sometimes conjugated with an enzyme (eg-hrp) to use for detection of biotinylated proteins.
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
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#3
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member
Posted 29 January 2010 - 06:13 PM
mdfenko, on Jan 29 2010, 07:00 AM, said:
you don't need to bind avidin to your protein to biotinylate it. you can biotinylate directly.
besides, avidin is relatively large (even monomeric) and may interfere with protein function.
avidin is often immobilized on a resin and is sometimes conjugated with an enzyme (eg-hrp) to use for detection of biotinylated proteins.
besides, avidin is relatively large (even monomeric) and may interfere with protein function.
avidin is often immobilized on a resin and is sometimes conjugated with an enzyme (eg-hrp) to use for detection of biotinylated proteins.
Thanks! Maybe I should state my problem more clearly. I want to conjugate protein A with a biotinylated ssDNA. I plan to fuse protein A with monomeric avidin.(Of course, I will introduce a linker peptide between protein A and the avidin to help the two fold correctly.) The avidin will mediate the coupling of protein A with the ssDNA. Now my worry is, can the moderate affinity (Kd~100 nM) of monomeric avidin and biotin provide stabel coupling for protein A and ssDNA?
#4
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an elder
Posted 01 February 2010 - 07:23 AM
fallmonkey, on Jan 29 2010, 09:13 PM, said:
Now my worry is, can the moderate affinity (Kd~100 nM) of monomeric avidin and biotin provide stable coupling for protein A and ssDNA?
that depends on the conditions that you treat the complex. check the data sheet to determine binding and releasing conditions. avoid the releasing conditions and the link should be stable.
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
i do what i get paid to do
