The first method is silanization of PDMS surface with 50% v/v in ethanol followed by treatment with Glutaraldehyde which presents a site for antibody binding.
In the second method BSA is passively adsorbed on the surface. Then Protein A is coupled with BSA with activation using GA. This presents a site for covalent binding of antibody to PDMS surface.
There is no concluding literature which shows which method is better. Different research groups have shown both the methods to be better than the other, hence contrasting data and the confusion.
I would appreciate if somebody can suggest which is the better method?
Hi Rohan, I don't understand why you chose chemistries that you describe.
You treat PDMS surfaces with some silane. I assume that the silane you are using is amino functionalized trimethoxysilane. Then you treat this surface with glutaraldehyde?
Bad idea. Glutaraldehyde has bifunctional aldehyde groups that will crosslink the amine functional groups of the silanes leaving very few free reactive amine groups.
Using BSA absorbed to a surface as a stable platform is a bad idea. BSA can be washed away because it is not covalently bound to the surface.
If it were up to me, I would coat the surface with trimethoxysilane with epoxy groups and then incubate them with purified protein A or Protein G or purified antibodies in non-primary amine buffers.
On glass or quartz we had very good results with glutaraldehyde. I would prefer a combination of your two approaches:
1. Aminosilanization
2. Glutaraldehyde
3. Protein G
4. Antibody
The method of Steingrimur is also very good. However, it needs a relatively high concentration and long incubation of protein A/G due to the low reactivity of epoxy groups.
Hi Michael, the reaction of epoxide with proteins can be increased 50-100 fold within 30-60 minutes by a simple, inexpensive and obvious procedure.
Can you guess what it is?
Thank you Steingrimur and Micheal for your answers. I have ordered both APTES and Protein G, and will try both the methods suggested by you.
@ Steingrimur, I also thought that BSA adsorption was not a good idea for the same reason, but a number of people have claimed it to be quite useful, and hence the question.
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