Guidelines
SURFACE PLASMON RESONANCE
Purity/quality of the sample needed
Purity and homogeneity of analytes has to be exceptional. Proteins, for example, have to be tested with the SDS-PAGE with minimum of 1 mg protein loaded, where they should appear as a single well visible band (e. g. at least 95 % purity). Similarly, DNA has to be tested on agarose gel and lipid vesicles have to be tested with DLS for their homogeneity.
Concentration of the analyte needs to be determined as accurately as possible (spectrophotometrically) and it should be measured right before the experiment.
Purity of the ligand (molecule attached to the sensor chip) can be lower.
Amount needed for a typical measurement
Depends on the biomolecule and interaction characteristics.
Ligand (attached to the surface): ~ 10-50 µg of a protein/~0.1-0.3 µg of a DNA/~ 100–300 µl of 200 µM vesicles for one immobilization.
Analyte: ~200 µl of a biomolecule with a concentration around 10 x expected KD (per one titration).
Other requirements
Guest researchers are kindly asked to fill in the Sample request form regarding sample quality, experimental conditions and plausible previous results.
Buffer considerations
Buffers should be filtered through 0.22 μm pores and, if possible, contain 0.005 % P-20 surfactant (Tween-20).
Most commonly used buffers are HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005 % P20), TBS-P (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.005 % P20) and PBS-P (10.1 mM Na2PO4, 1.8 mM KH2PO4 pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.005 % P20).
Additives to suppress non-specific binding are often added to buffers: 0.1 % BSA, 0.1–10 mg/ml CM-dextran (in case of dextran sensor chips), raising the detergent concentration (up to 0.1 %), raising the salt concentration (up to 250 mM NaCl).
When working with lipid vesicles, buffers should not contain detergents.
Tris-containing buffers should be avoided in the amine-coupling (ligand immobilization) step.
When samples require glycerol or DMSO, special care should be taken to match ligand and reference surface.
Concentrated organic solvents as well as long-term exposure to extreme pH (<3 and >11) should be avoided. The table below summarizes the chemical resistance of the microfluidic system.
|
Solution |
Concentration |
Compatibility |
|
acetonitrile |
50 % |
max 10 min injection |
|
dimethyl formamide (DMF) |
50 % |
max 10 min injection |
|
dimethyl sulphoxide (DMSO) |
50 % 10 % |
max 10 min injection long-term (as a running buffer) |
|
ethanol |
70 % 10 % |
max 10 min injection long-term (as a running buffer) |
|
ethylene glycol |
100 % |
max 10 min injection |
|
formic acid |
70 % |
max 10 min injection |
|
formamide |
40 % |
long-term (as a running buffer) |
How to improve 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 and 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.
Reagents 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
|
Useful links
SPR
SPR Pages: author Arnoud Marquart, everything about an SPR and biomolecular interactions.
Biacore: website of the Biacore company (technology description, protocols, applications, SPR news).
Biaffin: biomolecular interaction analysis service.
Bisensor tools: D. G. Myszka group. Protocols, analysis of results, publications.
MICROSCALE THERMOPHORESIS
ISOTHERMAL TITRATION CALORIMETRY
PRACTICAL INFORMATION FOR GUEST RESEARCHERS
Travel to Ljubljana
If you come to Slovenia by plane, there are busses and cabs from the airport to Ljubljana (approx. 20 min drive).
Accommodation in Ljubljana
|
Hotel |
Distance to the TNA site |
Hotel rating |
approx. price person/night |
contact |
|
InterContinental |
30 min walk or 6 min by bus No. 6 |
***** |
120 € |
|
|
Hotel Cubo |
15 min walk or 12 min by bus No. 1+6/9 |
**** |
100 € |
|
|
Hotel Lev |
25 min walk or 20 min by bus No. 9/1 |
**** |
65 € |
|
|
Atelier Hotel |
25 min walk or 20 min by bus No. 9/6/1 |
*** |
50 € |
|
|
B & B Vila Teslova |
1 min walk |
*** |
70 € |
|
|
Kavon Apartments |
6 min walk |
*** |
110 € (appropriate if family or friends are joining) |
|
|
Fine Ljubljana Apartments |
11 min walk |
*** |
120 € (appropriate if family or friends are joining) |
|
|
Hotel Mrak |
12 min walk |
*** |
115 € |
|
|
Hotel Castle View Fortuna |
17 min walk |
|
30 € |
|
|
Hostel Dežnik |
20 min walk or 20 min by bus No. 1+6/9 |
|
35 € |
Local travel in Ljubljana
Both the National Institute of Chemistry and the Department of Biology are in the walking distance from the city centre (15 min). However, there are local buses (LPP) and rent-a-bike stations (BicikeLJ) throughout the city, and there is a unified card or mobile application (Urbana) needed for their usage.
Administrative procedures for carrying out experimental work on the site
Guest researchers arrange the accommodation themselves.
Guest researchers that will be performing experiments on site are asked to get acquainted with rules regarding work with GMOs (confirmed with a signature at doc. dr. Nada Kraševec) and working safety (confirmed with a signature at dr. Andreja Drolc) and to fill in the following forms:
- Guest researcher announcement (Najava obiska zunanjega sodelavca),
- Statement for guest researchers (Izjava za zunanje sodelavce),
- Pisni sporazum – skupno delovišče (only for Slovenian researchers), and
- GMO risk assessment addendum (Dodatek k oceni tveganja), if there are any novel GMOs brought to the site.
In need of assistance, contact neza.omersa@ki.si.
Other relevant information for on-site experimental work at the National Institute of Chemistry
Entrance to the NIC is from Langusova street. The guest gets the permission card at the entrance and the card allows access to the specified infrastructure.
Other infrastructures for measuring molecular interactions
Molecular interactions consortium is a partner of MOSBRI. Whether in need of the infrastructure that is not available here, you can also apply to perform experiments at 14 other TNA (trans-national access) sites of MOSBRI partners from Europe.