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

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 toolsD. G. Myszka group, University of Utah, ZDA. Protocols, analysis of results, publications.


MICROSCALE THERMOPHORESIS


Nanotemper


ISOTHERMAL TITRATION CALORIMETRY


 

 

PRACTICAL INFORMATION FOR GUEST RESEARCHERS


Travel to Ljubljana

If you come to Slovenia by airplane, there are buses and taxis from the airport to Ljubljana (approx. 20 min ride).

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 €

Booking.com

Hotel Cubo

15 min walk or

12 min by bus No. 1+6/9

****

100 €

Booking.com

Hotel Lev

25 min walk or

20 min by bus No. 9/1

****

65 €

Booking.com

 

Atelier Hotel

25 min walk or

20 min by bus No. 9/6/1

***

50 €

Booking.com

B & B Vila Teslova

1 min walk

***

70 €

Booking.com

Kavon Apartments

6 min walk

***

110 € (appropriate if family or friends are joining)

Booking.com

Fine Ljubljana Apartments

11 min walk

***

120 € (appropriate if family or friends are joining)

Booking.com

 

Hotel Mrak

12 min walk

***

115 €

Booking.com

Hotel Castle View Fortuna

17 min walk

 

30 €

Booking.com

Hostel Dežnik

20 min walk or

20 min by bus No. 1+6/9

 

35 €

Booking.com

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:

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.