GFP visibility vs concentration?

[kenny kostenbader]

Hi All,

I was wondering how much GFP (in aqueous solution) would be required to glow bright enough to see with the naked eye? Or does it depend more on the intensity/color of the excitation lighting?

Thanks!

Kenny

[Nathan McCorkle]

you might try finding the dosage (watts of light per area... i.e.
w/cm^2) that photobleaching of GFP occurs at, then from there
determine the maximum fluorescence (watts of light per area, or # of
photons), then find the minimum detection limit for the eye for that
wavelength (watts/area or photons).

With that you should be able to determine how much light a single GFP
gives off at full brightness, then you can divide the minimum
sensitivity of the eye by that number to get # of GFPs needed. You
could then modify the equations with lower input power, in case you
don't have a super-bright UV light that is capable of photo-bleaching.

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--
-Nathan

[Avery louie]

or you could get some GFP and serially dilute it until you cant see it.

Filters help block the light that causes the excitement, but also make the overall image dimmer.

How do you measure GFPs/volume?

--A

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[kenny kostenbader]

Nathan, remind me to come back to you when I have a scientific heavy-lifting problem on my hands :) that's quite the solution!

Avery, I'd be able to get a value for mg/ml of protein from an absorbance reading.

I was hoping to find a set of pictures that compare jars of GFP with differing concentrations, something like that. When I get to my own samples, I'd be happy to perform some dilutions and post pictures somewhere. Someone out there will find that interesting :)

Kenny

[Nathan McCorkle]

Based on this:
http://www.ecse.rpi.edu/~schubert/Light-Emitting-Diodes-dot-org/Sample-Chapter.pdf

and the data on quantum yield
(http://en.wikipedia.org/wiki/Fluorescence#Quantum_yield) here
http://en.wikipedia.org/wiki/Green_fluorescent_protein#GFP_derivatives

you should be able to determine what you want, or be very close to
having all the pieces to get what you want.

On Thu, Jan 16, 2014 at 7:35 PM, kenny kostenbader

> https://groups.google.com/d/msgid/diybio/0eeeb02b-706c-4c33-9b58-fdb456961fbc%40googlegroups.com.

[Cathal Garvey]

IIRC, protein absorption is measured at 280nm? Make sure that's not in a
significant absorption band for your GFP fluorophore, or the fluorophore
might throw off your readings.

That is, I don't think the 280 readings generally assume that the
protein content of the solution has an unusually high absorbance at that
wavelength, but a GFP variant might! :)

On 17/01/14 03:35, kenny kostenbader wrote:
> Nathan, remind me to come back to you when I have a scientific
> heavy-lifting problem on my hands :) that's quite the solution!
>
> Avery, I'd be able to get a value for mg/ml of protein from an absorbance
> reading.
>
> I was hoping to find a set of pictures that compare jars of GFP with
> differing concentrations, something like that. When I get to my own
> samples, I'd be happy to perform some dilutions and post pictures
> somewhere. Someone out there will find that interesting :)
>
>
> Kenny
>
>
> On Thursday, January 16, 2014 7:50:08 PM UTC-5, Avery wrote:
>>
>> or you could get some GFP and serially dilute it until you cant see it.
>>
>> Filters help block the light that causes the excitement, but also make the
>> overall image dimmer.
>>
>> How do you measure GFPs/volume?
>>
>> --A
>>
>>

>> On Thu, Jan 16, 2014 at 6:17 PM, Nathan McCorkle <nmz...@gmail.com<javascript:>

>>> wrote:
>>
>>> you might try finding the dosage (watts of light per area... i.e.
>>> w/cm^2) that photobleaching of GFP occurs at, then from there
>>> determine the maximum fluorescence (watts of light per area, or # of
>>> photons), then find the minimum detection limit for the eye for that
>>> wavelength (watts/area or photons).
>>>
>>> With that you should be able to determine how much light a single GFP
>>> gives off at full brightness, then you can divide the minimum
>>> sensitivity of the eye by that number to get # of GFPs needed. You
>>> could then modify the equations with lower input power, in case you
>>> don't have a super-bright UV light that is capable of photo-bleaching.
>>>
>>> On Thu, Jan 16, 2014 at 2:26 PM, kenny kostenbader
>>> <kenst...@gmail.com <javascript:>> wrote:
>>>> Hi All,
>>>>
>>>> I was wondering how much GFP (in aqueous solution) would be required to
>>> glow
>>>> bright enough to see with the naked eye? Or does it depend more on the
>>>> intensity/color of the excitation lighting?
>>>>
>>>>
>>>> Thanks!
>>>> Kenny
>>>>
>>>> --
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>>> Groups
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>>> diy...@googlegroups.com <javascript:>.

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>>> this group at
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>>>> Learn more at www.diybio.org
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>>>> To view this discussion on the web visit
>>>>
>>> https://groups.google.com/d/msgid/diybio/639d9629-78b2-4268-83be-4c04e8757de1%40googlegroups.com
>>> .
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>>>
>>>
>>>
>>> --
>>> -Nathan
>>>
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>>> Visit this group at http://groups.google.com/group/diybio.
>>> To view this discussion on the web visit

>>> https://groups.google.com/d/msgid/diybio/CA%2B82U9L3w%3DtFYKsV26y4gdg04Te6fiVXFW0U%3Dz8fZR_9NBS-MQ%40mail.gmail.com
>>> .

[Nathan McCorkle]

Or a Bradford or Lowry type assay:
http://en.wikipedia.org/wiki/Bradford_protein_assay
http://en.wikipedia.org/wiki/Lowry_protein_assay

--
-Nathan