Useful

SURFACE PLASMON RESONANCE


 

How much ligand do I need?

For an average immobilization you need 10-50 µg of proteins.

How much analyte do I need?
This depends on the affinity between the ligand and analyte. 

How to improve the data quality?

Cause of deviation
Possible solution
Samples are chemically or conformationally impure
Purify proteins
Samples self associate or aggregate
Check proteins using analytical centrifugation
Multivalent interactions/avidity effects
Keep monovalent ligand in solution
Chemical heterogenity in ligand imposed by immobilization procedure
Orient the immobilization of the ligand or use a capture step
Mass transport effects/rebinding
Use low surface densities anf high flow rates
Matrix effects, bulk refractive index changes, instrument drift and injection noise
Match sample and running buffers and set up a reference cell
Crowding/steric hindrance
Use low surface densities
Nonspecific binding of analyte to surface
Use reference cell
pH or temperature effects
Equilibrate sample and running buffer
Experimental noise
Replicate and randomize samples
Complex binding mechanism
Use numerical integration and global data analysis

(Myszka, D. G. (1997) Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors, Current Opinion in Biotechnology, 8:50-57), PubMed

Flowrate and concentration requirements

Guide to surface binding capacities and flow rates for different interaction experiments (Myszka, D. G.; 1997) Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors, Current Opinion in Biotechnology, 8:50-57), PubMed.

 

Regenets for chip regeneration

Agents which may be used in regeneration of the sensor chip surface after binding of the analyte.

Agent
CM5
L1
SA
AU
Acetonitrile
20%
20%
20%
20%
DMSO
10%
10%
8%
8%
DTE in HBS-buffer
0.1 M
0.1 M
10 mM
10 mM
EDTA
0.35 M
0.35 M
-
0.35 M
Ethanol
70%
70%
70%
70%
Ethanolamine
1 M
1 M
100 mM
100 mM
Ethylene glycol
100%
100%
50%
50% (in HBS buffer)
Formamide
40%
40%
40%
40%
Formic acid
20%
20%
20%
20%
Glycine pH 1.5-3.0
100 mM
100 mM
10 mM
100 mM (pH 2.3)
HCl
100 mM
100 mM
-
100 mM
Imidazole
300 mM
300 mM
-
300 mM
MgCl2
4 M
4 M
4 M
4 M
NaOH
100 mM
100 mM
100 mM
100 mM
NaCl
5 M
5 M
1 M
1 M
SDS
0.5%
0.5%
5%
0.5%
Surfactant P20
5%
5%
-
5%
Urea
8 M
8 M
8 M
8 M