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How To Prepare A Successful Feasibility Study
How To Prepare A Successful Feasibility Study
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.
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ConfoCor 2
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ConfoCor 2
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 .................................................................
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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
Antigen ⇔ Antibody
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ConfoCor 2
kinetic constants
binding constants
degree of binding
viscosity
other parameters:
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
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ConfoCor 2
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 ................................................................................................
the following additives are used for the stabilisation of the solution:
............................................................................................................................................
............................................................................................................................................
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ConfoCor 2
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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
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.
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ConfoCor 2
Safety requirements
No special safety requirements are necessary.
The following safety requirements are necessary
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
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ConfoCor 2
For small molecules with a mass below 10 kD a mass spectrum is strongly recommended!
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.
the affinity of the ligand to the labeled partner has been determined, the Kd value is........
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ConfoCor 2
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Signature
Thank you very much for taking your time filling out the questionnaire.
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