Imagine I have an SPR chip, either biacore, or a custom gold surface, and I activate with EDC/NHS, add some protein for covalent immobilization, and then want to block out the remaining sites.
At this point, I have several options. First, I could use a quenching agent like ethanolamine to presumably block the remaining activated sites. Secondly, I could add a non-specific protein cocktail like cassein or protein sera to take up those spots. Or I could use a combo?
My goal is to have a surface that's very non-reactive and neutral. I worry that a quenching agent may still leave my surface net-charged, resulting in surface-protein interaction, but the other proteins deposited on the fiber could result in more non-specific protein-protein interactions.
We have studied several blocking agents for antibody binding using QCM-D and the best performing is the detergent tween-20. But this may vary depending on the type of proteins and its size. You can test several of them by using SPR, following over time the adsorption (if any) and desorption (if any) signal over your protein layer.
You have yet another option - wait, because EDC/NHS activation is not stable. So simply wash with PBS buffer for about an hour after immobilisation. We observe reproducible antibodies adsorption/desorption(regeneration) cycles after that and for me it is the best confirmation. I do not have an exact reference for this tip, but it is said to be so at least in the following article of Whitesides' group:
L. Joydeep, L. Isaacs, J. Tien and G.M. Whitesides. “A Strategy for the Generation of Surfaces Presenting Ligands for Studies of Binding Based on an Active Ester as a Common Reactive Intermediate: A Surface Plasmon Resonance Study.” Anal.Chem. 71, no. 4 (February 1999): 777–90. doi:10.1021/ac980959t.
Marite,
Can you clarify how tween20 works in this application? It is a detergent/emulsifying agent, no? Wouldn't it damage the proteins that you have covalently immobilized prior to quenching?
Shavat,
Thanks. If you let the reactive intermediate return to it's original COO- group this way, then your surface will have an excess of negative charges. We've observed that such a surface, even with some covalently-bound ligand, will have significant non-specific binding. Thus, how do you know that your reaction is specific? If you add other proteins like milk proteins and so forth, do you not see any effect?
Hi Adam,
I guess you are using self-assembled monolayers (SAMs) with COO- group and then covalently conjugate proteins to the SAMs. What molecules are you using and how are you checking the non-specific binding?
To reduce non-specific binding, blocking with other protein like BSA do help a lot. (But this may also affect the protein bindings afterwards.) If you have PEG SAMs plus BSA blocking, I guess you are much safer.
Do you mean you use tween20 in your running buffers, or that you literally block with it?
If you are concerned about the negative charge on the dextran surface, you can block with ethylene diamine instead of ethanolamine to help neutralize the negative charge. Additionally, using Tween 20 in the running buffer along with BSA and even dextran will help a lot with non-specific binding.
Hi Phillip, thank you. We are stuck, and your recommendation i think might be helpful.
Could you share a protocol, please?
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