A Sensorgram


A sensorgram

In the previous pages you have learnt what the important parts are of the curves and sensorgrams. The following figures show a low and a high-quality sensorgram.

Low quality sensorgram
Low quality sensorgram

High quality sensorgram
High quality sensorgram

In conclusion: what to look for before fitting a set of curves?

  • The baseline is flat. When the running buffer is flowing, the drift of the baseline is close to zero.
  • On injection of the analyte, the buffer jump is very low.
  • The association is free of mass transport.
  • The curves are following the single exponential
  • The curves have sufficient curvature
  • There is at least one replicate
  • The analyte concentration is between 0.1 – 10 times the expected KD
  • The curves are well spaced in the sensorgram
  • At least one of the curves is reaching steady state (when possible)
  • The overall response is low
  • The response is proportional to the analyte concentration and kinetics
  • The dissociation is long enough for a sufficient decay in response
  • The injections are randomized to avoid systematic errors

When the curves are not of high quality, optimize the experimental conditions before attempting any fitting. Fitting on bad curves will give bad answers and waste your time. Therefore, start optimizing (1).

Stes that can be taken:

  • check ligand and analyte for purity and uniformity
  • lower ligand density on the sensor chip
  • increase flow rate to check for mass transport
  • reverse ligand and analyte on the sensor chip
  • change the immobilization chemistry (e.g. thiol coupling)
  • add or remove salts
  • add or remove detergents

References

(1)Rich, R. L. and Myszka, D. G. Survey of the year 2007 commercial optical biosensor literature. J.Mol.Recognit. 21: 355-400; (2008). Goto reference

Comments  

# cristi 2013-01-04 20:18
i am new to SPR, soo if the question is too foolish please understand.
At the conclusion for what to look at when fitting, you say that the overall response is low, is this valid only for the picture above or for small molecules(LMW).
If we had an analyte of lets say 50 kDa wouldn't the response be bigger?
Reply | Reply with quote | Quote
# Arnoud 2013-01-04 20:19
Depending on the size of the ligand and analyte the response will be low or higher.
The take away message is: a low response is better. This means that when you have large molecules you have to immobilize less
Reply | Reply with quote | Quote
# safia 2013-03-21 20:20
:-| Rich, R. L. and Myszka, D. G. Survey of the year 2007 commercial optical biosensor literature. J.Mol.Recognit. 21: 355-400; (2008).
Reply | Reply with quote | Quote