Instant decrease of counts after adding second component
I am trying to see if I can measure the weak interaction of the surface of an enzyme with different polymers with various side groups with the MP.
I have found the concentration of polymers that doesn't create much of background noise.
What I have been doing is to first add a certain amount of the enzyme into the drop (1uL), do a measurement, and then add polymer (1 uL-2uL) to make a total of about 20uL.
I've noticed that as soon as the polymer is added, the mass only changes slightly, but the counts drop significantly (sometimes half).
If I add more of the polymer solution, the count drops even more, and I don't think this is due to dilution.
Concurrently, the unbinding events seem to increase.
Would it be possible that it's because the presence of polymers are lowering the affinity of the enzyme to the glass surface?
Hi Hye,
First of all, it is normal to experience a decrease in counts if a sample continuous to be recorded several minutes after having been added on the glass surface. The proteins will land on the glass and typically stay there, decreasing the number of molecules in solution available to produce new landing events. As a general recommendation, we would encourage you to mix the enzyme with the polymer separately in a tube, then pipette the mix on the glass and record it. This applies to the study of any interaction, or the mixing of any new component: it is usually better to add the new component separately in a tube and record the mix in a new well.
As to whether the polymer might be having an effect on the interaction of the protein with the glass surface, that is, indeed, an option. Maybe the polymer is coating the protein and impeding its interaction with the glass, or maybe the polymer is directly coating the glass surface, or could be something else. You could try coating the glass with Poly-L-Lysine to apply a layer of charges on the glass that will promote different surface interactions – there is a protocol in our YouTube channel about that and it only takes a couple of minutes.
Hope this helps!