I am using ez-link biotin from pierce (http://www.piercenet.com/product/ez-link-nhs-biotin), and noticed they offer different length spacer arms for different level of steric hinderance reduction.
The thing is, I have no real sense of how long is enough, what considerations one should take into account, and so forth for my needs. I bascially want to biotinylate proteins to neutravidin, which I've deposited on a gold surface. The question is how much spacer arm do I need? And what considerations are going to manifest as first-order?
Thanks
Based on my experience, I think one LC spacer is enough to reduce the steric hindrance. In fact, you can see from the products list of pierce that the length of different spacers is similar, expect the PEG12 spacer. The difference between LC spacer and PEG spacer is the water solubility, and some researchers think that PEG spacer can also reduce the nonspecific absorption of biotin to protein. So, I think you can just take the choice as you like.
Long space arm could reduce the steric hindrance, which is good for biotinylated proteins to bind to avidin and maintain their functionality; but since avidin only has 2 or 4 binding sites for biotins and that each protein may be conjugated with multiple biotins, long space arm probably increase the change that multiple avdins are blocked by biotins on one protein, resulting in low efficiency of binding biotinylated protein to avidin. I once worked with SH-PEG-Biotin SAM, I could attached streptavidin to the SAM, but not biotinylated protein to streptavidin, because the peg chain was too long.
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