How do I coat glass with streptavidin? (a written protocol would be nice if available)
I'm trying to make a system that can detect a biotin based marker in an ELISA like setup, however, I can't use standard plates due to the constraints of my system so I have to use glass... specifically fisher microscope slide glass. Is there any straightforward protocol for attaching streptavidin to glass that is ideally more than just from electrostatic interactions? I'd really like it to be fairly uniform as well and bind well if possible.
1-You can do this by immerison of the glass substrate in a solution containing 5mg/ml of streptavidin in PBS or tris buffer at pH7. Streptavidin would adsorb on it. But the coverage is not perfectly uniform.
2-Another alternative is to derivatize the glass with amine groups. This could be done by immersing the glass for 20 min in 1Molar NaOH(aq), rinsing, then submerge the glass in a methanol solution of 3-aminepropyl-trimethoxy silane. Rinse with methanol. Rinse with water. Then submerse the amine modifed glass in streptavidin solution.
3-Yet another way, based on the fact that albumin adsorbs very well and coats quite uniformly many hydrophylic surfaces: You can prepare 10mg/ml of biotinylated-albumin in 1 ml of PBS buffer of pH=7. Then, submerse the glass in the solution. This will expose a number of biotine moieties which strongly bind to streptavidin. Thus you form a layer of streptavidin on top of the albumin. SInce streptavidin has 4 sites, you will still have biotin binting sites on the streptavidin coated glass.
I have used methods 1 and 3 except that i mixed biotinilated albumin with regular albumin in a 1:10 proportion to get less streptavidin coverage:
http://www.pnas.org/content/102/30/10505.full.pdf
I hope this gets you started.
Dear Jose,
Could the same protocol be used if you are using a metal surface ?
Would appreciate your advice.
For metal surface, the best technique is using SAMs which contain biotin to bind streptavidin.
Hello Jose,
Jose H Hodak Alternative 2 sounds promising. Could you please refer a paper where I can find the protocol for derivitization of glass with amine followed by streptavidin functionalization. Thanks in advance.
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