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Hey guys I want to ask why I can't get any binding interaction if I use TRIS-NaCl buffer for protein quantification using aptamer as its bioreceptor. But I can see the binding interaction if I use PBS buffer The procedure step for my experiment :

1.) Self Assembled Monolayer (24-48 hrs) - NHS/EDC Activation (wash with acetate) - Streptavidin (Acetate buffer) - wash with acetate - wash with TRIS - ETH Blocking - wash with TRIS - Aptamer biotinylated (TRIS buffer) - Protein (TRIS buffer)

all washing buffer are using TRIS or PBS

And down below (my other method) I tried to exchange the buffer from PBS to TRIS after aptamer injection

2.) Self Assembled Monolayer (24-48 hrs) - NHS/EDC Activation (wash with acetate) - Streptavidin (Acetate buffer) - wash with acetate - wash with PBS - ETH Blocking - wash with PBS - Aptamer biotinylated (PBS buffer) - Buffer exchange (PBS to TRIS) - Protein (TRIS buffer)

I can see my aptamer signal in 1st and 2nd method, but still no binding occurred between my aptamer and my protein in TRIS buffer.

NexusRay
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    I don’t quite understand your protocol, but EDC/NHS activated carboxylates react with primary amines. Tris contains a primary amine and will be present at a much higher concentration compared to streptavidin. – canadianer Jan 17 '21 at 14:41
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    Are the pH and ionic strength of the two buffers the same? – canadianer Jan 17 '21 at 16:20

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I presume the aptamer is biotinylated; if not, that’s a likely reason for the outcome.

Have you used a biotinylated positive control to confirm the surface is functional with PBS? I understand you want to buffer exchange to Tris, but this positive control in PBS buffer is important.

Amine coupling involves covalent attachment of the 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to carboxylate on the chip surface, followed by replacement of this adduct with N-hydroxysuccinimide (NHS), thus creating an active surface to crosslink primary amines at slightly acidic pH. Then the unreacted active surface is quenched with ETH. Residual active surface would react with Tris that contains a primary amine.

Perhaps the ethanolamine blocking agent may not have completely inactivated the active surface following streptavidin amine coupling, enabling crosslinking of Tris buffer to that surface and impacting the ‘Protein’ capture?

Other possible reasons: Binding of the aptamer to streptavidin may sterically hinder binding of the 'Protein.' Also, the loss of the proper secondary structure of the aptamer may impact the 'Protein' binding. Perhaps refolding the aptamer prior to streptavidin capture may help?

Edit: Differences in pH and ionic strength may impact 'Protein' binding to aptamer when tested in Tris versus PBS buffers.

z1273
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  • Yes, the aptamer is biotinylated thus it can bind with the streptavidin. Yes and I also know that NHS ester will be released because of the primary amine of the TRIS buffer and create covalent amide bond.

    But I'm pretty sure right now that my ethanolamine blocking agent is deactivated all the NHS ester residue because I also double-check using BSA blocking to my SPR surface. That's why I'm pretty confused why with PBS my aptamer can catch the protein and with TRIS can't

     – NexusRay Jan 17 '21 at 15:11
  • It looks like the ethanolamine quenching may not have been sufficiently effective? – z1273 Jan 17 '21 at 15:12
  • Oh I forgot to mention that I can see my aptamer signal, but when I inject my protein I can't see its binding interaction and the signal keep going flat (not indicating any changes at all) – NexusRay Jan 17 '21 at 15:16
  • OK. Increased RU counts is a good sign. Has this lot of protein been confirmed to bind this lot of aptamer by a different technique? – z1273 Jan 17 '21 at 15:19
  • I got this aptamer and its protein from my co-worker and he said he already test and confirm it with ELISA. – NexusRay Jan 17 '21 at 15:22
  • But why I can see the binding interaction of my protein and its aptamer using PBS buffer? – NexusRay Jan 17 '21 at 15:33
  • I presume you have a control surface with no streptavidin, but everything else to confirm the ethanolamine quenched the residual active surface? Also, is the positive binding of the protein to its aptamer using PBS show reasonable dissociation rate constants? – z1273 Jan 17 '21 at 15:37
  • Yes, and i see reasonable association and dissociation rate constants of different kind of concentrations – NexusRay Jan 17 '21 at 15:41
  • At what pH was the 'Protein' binding done in the PBS and Tris experiments? – z1273 Jan 17 '21 at 16:01
  • Ah yes maybe because the pH and ionic strength difference, so because of that my aptamer cant bind with my protein properly even though my aptamer can bind with streptavidin. – NexusRay Jan 18 '21 at 03:03