how to prepare a successful feasibility study

8/14/2019 How to Prepare a Successful Feasibility Study

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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:

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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 cellstype 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

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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: ..............................



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: ..............................









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 crosscorrelation 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













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: ....................................................................

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

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

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

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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

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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%


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


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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 WiegräbeProduct Manager Advanced Imaging MicroscopyBusiness Group Microscopy

Carl Zeiss Jena GmbHCarl Zeiss GroupCarl-Zeiss-Promenade 1007745 JenaGermany

Phone: ++49 (3641) 64-3420Fax: ++49 (3641) 64-3144e-mail: [email protected]

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how to prepare a successful feasibility study

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