I am trying to perform covalent immobilization of streptavidin protein on glass surface. This is done by first coating the glass with 2% APTES-toluene and then with 2.5% glutaraldehyde-water solution. Then I add the streptavidin which is in PBS (pH 7.4) on the glass coverslip. This doesn't seem to work. Please let me know the right pH, salt and temperature conditions that are appropriate for the reaction between glutaraldehyde and amine groups created by APTES coating on glass.
Try increasing the pH for attachment. Are you including a reduction step?
7/5/17
Dear Sumanth,
If I were doing this, I'd use an alkaline pH. APTES has a primary amine in its structure. Glutaraldehyde reacts with this, forming a Schiff base. The carbonyl carbon of glutaraldehyde has a slight positive (+) charge owing to a dipole effect, so in the reaction w/ an amine, it is looking for something with a negative charge, such as the 2 electrons on the amino nitrogen. At a low pH, the electrons snatch a proton from water, forming a protonated amine. Since the electrons are "tied up" with the proton, the reaction with the carbonyl carbon of glutaraldehyde will be hindered. Raising the pH into the alkaline range will remove the proton, making the electrons available to react with glutaraldehyde.
See the attached reference.
I hope this information helps you.
Bill Colonna Center for Crops Utilization Research, Iowa State University, Ames, Iowa, USA [email protected]
Dear Omar,
What do you mean by a reduction step?
Dear Colonna,
Please check my protocol and suggest any changes.
My protocol goes as follows:
0. Cleaned in 5% HCl-MilliQ for 3hrs, rinsed with MilliQ
1. Piranha cleaning of coverslips at 120degC for 20min
2. Washing with MilliQ, dry under nitrogen
3. Oxygen plasma cleaning at 20W for 2min @ 250mtorr
4. Incubate them in 2% APTES-toluene at 60degC for 1hr
5. Sonicate them in toluene followed by water for 5min each, dry them under nitrogen
6. Cure them in hot air oven at 100degC for 2hrs
7. Store them in vacuum desiccator for 12hrs (so that I can start experiment on next morning)
8. Incubate them in 2.5% glutaraldehyde-MilliQ, pH~6 (I also tried 10% glutaraldehyde-10X PBS, pH 7.4) for 1hr at 25degC
9. Wash them in water, 1X PBS
10. Incubate them in 1mg/ml Streptavidin (diluted with MilliQ from powder form) for 3hrs
11. Then performed experiments with PEG-Biotin coated AFM tips in 1X PBS (pH 7.4), 10X PBS (pH 7.4), MilliQ (pH~6)
The reaction between an aldehyde and an amine is reversible, you need to use a reductant to produce an irreversible linkage. From your protocol, streptavidin should be dissolved in a proper buffer.
Dear Omar,
Please suggest me a reductant that I can use for my requirement.
And I have one question:
Glutaraldehyde has two aldehyde groups on either sides of the carbon chain.
Is it possible that these two aldehydes form bonds with amine groups on the glass surface? Perhaps then the protein cannot find free aldehyde groups to bind?
Please let me know your opinion.
I found that Sodium cyano borohydride would act like a reducing agent in this reaction.
Use cyanoborohydride as stated. For the other question, that is why you are supposed to use glutaraldehyde in excess.
In excess with respect to the number of amino groups on the surface
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