ConfoCor 2

How to prepare a successful feasibility study / evaluation

Dear customer,

we have made the experience that it will be very important for good FCS results to know as
much as possible about your samples and their preparation. With this questionnaire we
would like to help you to find the best way to do your experiment.

Title, name, first name ...................................................................................................

Function ...................................................................................................
Department / institute ...................................................................................................
Company, university etc. ...................................................................................................
Street ...................................................................................................
Zip code / city ...................................................................................................
Country ...................................................................................................
Telephone number ...................................................................................................
Telefax number ...................................................................................................
e-mail ...................................................................................................
WWW-adress ....................................................................................................

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 ConfoCor 2

1. General information about the assay which is planned to be measured

Background information on the system used:
...................................................................................................................................... ..
.................................................................................................................................... ...........
...........................................................................................................................
......................................................................................................................................
......................................................................................................................................
......................................................................................................................................

Short description of the analytical task:

......................................................................................................................................
......................................................................................................................................
......................................................................................................................................
......................................................................................................................................
......................................................................................................................................
......................................................................................................................................
......................................................................................................................................

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 ConfoCor 2

2. Information related to the classification of the interaction

 Receptor ⇔ Ligand
Information about the receptor
 solubilized  non-solubilized

 at vesicles
type of cell .........................................................................
receptor density .................................................................
size / diameter ....................................................................
homogeneity of the vesicles ...............................................

 homogenate
type of cell .........................................................................
receptor density .................................................................

 whole cells
type of cell ..........................................................................
receptor density .................................................................

Information about the ligand

 small organic molecule with a molecular mass of ............................................................
 peptide with a molecular mass of .....................................................................................

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 ConfoCor 2

 DNA ⇔ Protein
 measurement in solution  measurement in cells / on cell membranes

 Protein ⇔ Protein
 measurement in solution  measurement in cells / on cell membranes

 Nucleic acid ⇔ Nucleic acid

 DNA ⇔ DNA:  in solution  in cells / on cell membranes
 DNA ⇔ RNA:  in solution  in cells / on cell membranes
 RNA ⇔ RNA:  in solution  in cells / on cell membranes

 Antigen ⇔ Antibody

 Measurement in cells / on cell membranes

Field of interest
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
 Others
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................

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 ConfoCor 2

3. Information about the parameters to be evaluated during the trial

 diffusion times / diffusion coefficients

 kinetic constants

 binding constants

 degree of binding

 size of the molecule

 viscosity

 other parameters:
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................

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 ConfoCor 2

4. Information already known about the assay to be measured

 concentration range typically

used ......................................................................................

 size and structure of the interaction partners

 spherical ⇒ with a diameter of ...........................................................................
 elliptical ⇒ relation length/height ..........................................................................
 rod shaped ⇒ relation length/height ..........................................................................
 the molecules tend to form dimers
 the molecules tend to form higher aggregates (oligomers)
 number of binding sites ..................................................................................................
 molecular weight of interaction partner 1 ............................of partner 2 ........................

 The following constants are known

 binding constants ...........................................................................................................
 on-rate .............................................  off-rate ...........................................................
 the above mentioned parameters have already been determined under comparable
conditions using following method ..................................................................................
............................................................................................................................................
 non-specific binding of the assay components is known
 non-specific binding of the dye
to ...................................................................................
has been observed

In order to be able to differentiate between the labeled ligand and the complex formed, a more than 8-
fold difference in molecular weight would be optimal. With lower differences the accuracy of the
measurement will decrease and/or the measurement time will be longer. You should in any case avoid
mass differences below three. The molecular weight of the unlabeled partner can be increased by
coupling non-reactive analogues to it. Otherwise, you have to label both partners and use
crosscorrelation.

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 ConfoCor 2

5. Assay parameters
 number of measurements per day ......................................................................................
 assay volume typically
used ................................................................................................

 type of buffer ......................................................................................................................

 pH value .........................................................................................................................
 contains % BSA at .........................................................................................................
 following detergents are
necessary ................................................................................
 auto-fluorescence is known at (nm)
...............................................................................
 the buffer is filtered under sterile conditions

 the following additives are used for the stabilisation of the solution:
............................................................................................................................................
............................................................................................................................................

 the viscosity of the solution is

..............................................................................................

 Yes - the interaction partners are stable under assay conditions

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 ConfoCor 2

6. Properties of the dyes used for labelling

Dye used to label the smaller interaction partner

 The smaller interaction partner can be labelled using the dye
named .................................
 As excitation wavelength can be used:
 458 nm peak absorption is at: ..............................
 488 nm peak absorption is at: ..............................
 514 nm peak absorption is at: ..............................
 543 nm peak absorption is at: ..............................
 633 nm peak absorption is at: ..............................
 other: ..............................................
 please find enclosed the absorption spectrum

 Emission can be measured at

 > 475 nm peak emission is: ..............................
 475 - 525 nm peak emission is: ..............................
 > 505 nm peak emission is: ..............................
 505 - 530 nm peak emission is: ..............................
 505 - 550 nm peak emission is: ..............................
 > 530 nm peak emission is: ..............................
 530 - 600 nm peak emission is: ..............................
 > 560 nm peak emission is: ..............................
 560 - 615 nm peak emission is: ..............................
 > 585 nm peak emission is: ..............................
 585 - 615 nm peak emission is: ..............................
 > 650 nm peak emission is: ..............................
 other peak emission is: ..............................
 please find enclosed the emission spectrum

 quenching effects have been analysed with following results

............................................................................................................................................
 bleaching effects have been analysed with following results
............................................................................................................................................

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 ConfoCor 2

 The ratio between bound and unbound dye has been determined to be
................../.....................
 As an alternative I can use the following
dyes: ....................................................................

Dye used to label the larger interaction partner (only necessary for cross
correlation experiments)
 The larger interaction partner can be labelled using the dye named .................................
 As excitation wavelength can be used:
 488 nm peak absorption is at: ..............................
 633 nm peak absorption is at: ..............................
 other: ..............................................
 please find enclosed the absorption spectrum
 Emission is can be measured at
 > 475 nm peak emission is: ..............................
 475 - 525 nm peak emission is: ..............................
 > 505 nm peak emission is: ..............................
 505 - 530 nm peak emission is: ..............................
 505 - 550 nm peak emission is: ..............................
 > 530 nm peak emission is: ..............................
 530 - 600 nm peak emission is: ..............................
 > 560 nm peak emission is: ..............................
 560 - 615 nm peak emission is: ..............................
 > 585 nm peak emission is: ..............................
 585 - 615 nm peak emission is: ..............................
 > 650 nm peak emission is: ..............................
 other: peak emission is: ..............................
 please find enclosed the emission spectrum

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 ConfoCor 2

 cross talk between the two dyes used for the smaller and the larger interaction partner
has been analysed with following results:
............................................................................................................................................
 quenching effects have been analysed with following results:
............................................................................................................................................
 bleaching effects have been analysed with following results:
............................................................................................................................................
 The ratio between bound and unbound dye has been determined to be
................../.....................
 As an alternative I can use the following
dyes: ....................................................................

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 ConfoCor 2

7. Information about the material needed to perform the study

 I will have the following amount of reaction partner 1 available for the feasibility
study: ....................................................................
 I will have the following amount of reaction partner 2 available for the feasibility
study: ....................................................................

Usually a total amount of 200 pmol of both reaction partners are recommended for a feasibility study,
for an extended study 1 nmol is preferable.

 I will bring all additional chemicals with me. They are

named: ....................................................................
....................................................................
....................................................................
....................................................................
....................................................................

 Special storage conditions for materials used:

 Storage temperature required:.....................................................................
 Storage temperature recommended:............................................................
 Material is air sensitive
 Material is light sensitive
 Material is shock sensitive
 Material specially packaged (e.g. oversized, heavy): ....................................
 Others:.................................................................................................
 if stored according to the recommendations the material will be stable at least
................................. weeks.

 Handling temperature during experiment:...................................................

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 ConfoCor 2

 Safety requirements
 No special safety requirements are necessary.
 The following safety requirements are necessary
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................

because the material used is

 toxic
 infectious
 inflammable
 caustic
 others: ...........................................................................................................

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 ConfoCor 2

8. Properties required for the labeled ligand

 Purity of the substance...................................................to be labeled
 the substance is available in homogeneous form
 impurities of..................% have been observed

Impurities should not exceed 10%!

 Purity of the labeled ligand

 identification has been performed by mass spectroscopy
 enclosed please find the related print out(s)

 ligands with a molecular mass ≤ 10 kD have to be purified by HPLC to ≥ 95%

 enclosed please find the related printout(s)

 ligands with a molecular mass ≥ 10 kD have to be purified by gel filtration or HPLC to ≥

90%
 enclosed please find the related printout(s)

For small molecules with a mass below 10 kD a mass spectrum is strongly recommended!

 Spectral properties of the labeled ligand

 spectra

 enclosed please find the absorbtion spectrum

 enclosed please find the fluorescence spectrum

 Quantum yield
 the quantum yield of the labeled ligand differs not more than 15% from that of the dye
used for labeling
 enclosed please find the related printout(s)

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 ConfoCor 2

 Quenching
 quenching effects have been analysed with the following results
............................................................................................................................................

The count rate per molecule (cpm) of the labeled ligand in the FCS measurements should be 10 times
higher than the background count rate of the buffer. A minimum cpm ≥ 20 kHz is sufficient.

 Thermodynamic properties of the labeled ligand

 the affinity of the ligand to the labeled partner has been determined, the Kd value is........

 the labeled ligand binds up to 100%

 there are no active isomers

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 ConfoCor 2

9. Additional information and comments

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Signature

Thank you very much for taking your time filling out the questionnaire.

Dr. Winfried Wiegräbe

Product Manager
Advanced Imaging Microscopy
Business Group Microscopy

Carl Zeiss Jena GmbH

Carl Zeiss Group
Carl-Zeiss-Promenade 10
07745 Jena
Germany

Phone: ++49 (3641) 64-3420

Fax: ++49 (3641) 64-3144
e-mail: wiegraebe@zeiss.de

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