Fluorescence Spectroscopy

 
 

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Instruments:


The EPPF currently houses two instruments with fluorometric capabilities:


A SpectraMax M5 multimode plate reader (located in lab 3.10).




A SPEX FluoroMax 3 (located in lab 3.5).




Please click here for individual platform/resource costings.

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Features of the M5 multimode plate reader:

  1. cuvette and 6, 12, 24, 48, 96, 384 well-plate format.


  1. five detection modes for plate based-assays - UV-Vis absorbance, fluorescence intensity, fluorescence polarization, time-resolved fluorescence, luminescence.


  1. three detection modes for cuvette based-assays - UV-Vis absorbance, fluorescence intensity luminescence.


  1. dual monochromator determines optimal excitation and emission settings.


  1. wavelength ranges: 200 - 1000 nm (absorbance) and 250 - 850 nm (fluorescence).


  1. temperature control from ambient + 2°C to + 60°C.


  1. endpoint, kinetic, spectrum, and area-well scanning read types.


  1. plate shaking capability.


  1. bottom-reading capability for fluorescence intensity, time-resolved fluorescence and luminescence.


  1. Well volumes ≤ 50 μL, ensuring low consumption of materials, including protein and ligands.


  1. Run times can be as short as 25 minutes allowing for high throughput assays.


  1. Wide range of ligand binding affinities can be detected.


  1. Versatile software SoftMax Pro for data acquisition, analysis and management and cross-plate analysis and custom calculations.


  1. Unique PathCheck technology available to measures the depth of samples in a microplate.


  1. Homogeneous and heterogeneous biochemical- or cell-based assays.


Technical specifications for the M5 multimode plate reader.

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Features of the FluoroMax3 spectrofluorometer:

  1. Ideal for high-resolution, sensitive fluorometric work (ca five times the sensitivity of the SpectraMax M5 Multimode plate reader).


  1. Ideal for intrinsic tryptophan and tyrosine fluorescence.


  1. Temperature of the sample block is controlled using a circulating water bath.


  1. Accommodates standard 10 mm path-length fluorescence cuvettes.


  1. Proprietary software, Fmax3*, is very easy to use; it controls the experimental set-up and data acquisition.


  1. Xenon arc lamp.


  1. Wavelength selection is achieved by the optical gratings of the excitation and emission monochromators.


  1. adjustable slits (adjustable in the software) further resolve light wavelengths.


* Note: this software is very old and has limited functionality with regard to data processing, we normally use this software only for data acquisition and viewing, and routinely copy the data collected directly into an Excel spreadsheet for processing/analysis.


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Sample prep/handling:

  1. 0.2 μm filtering is recommended as light scatter can be a problem.


  1. Sample solutions should be buffer-matched to any buffer controls.


  1. For titrations, corresponding buffer control titrations should be performed to take into account any fluorescence originating from the titrant.


  1. Cuvette must be thoroughly clean before and after use. Use lens tissue to clean the outer surface (remember dust particles scatter light!). Xenon arc lamp.


  1. Recommended volume for the standard cuvette is 0.5 ml (minimum volume is 350 μl although recommend using at least 400 μl).


  1. Typical protein concentrations are 1-10 μM (obviously this is molecule-dependent and is related to the environment, and proportion, of tryptophans and other aromatics/fluorophores. 


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Booking and rules for use of the SpectraMax M5 and FluoroMax 3:

  1. Contact details of facility staff for training and advice: Martin Wear, Liz Blackburn, Matt Nowicki, or Sandra Bruce.


  1. The plate reader must be booked prior to use; a web-based booking system is available for registered users at SpectraMax online booking.


  1. One half or full day can be booked at any one time. A single day is classified as 9 am until 8 am the following day.


  1. The instrument cannot normally be booked for use over the weekend.


  1. 24 hours notice should be given if a booked slot cannot be used. If the instrument is booked and not used, the booked user will be charged a a nominal fee (click here for details). The system administrators can delete booked user slots with 24 hours notice.


  1. Only the person booking the instrument should use the instrument within that booked period.


  1. Do not attempt to use this instrument if you don¡¦t know what you are doing; please contact one of the facility staff (listed above) for training and advice.


  1. If you have problems with the instrument at any time, contact one of the facility staff (listed above) immediately.


  1. DO NOT pipette into the plate/cuvette whilst it is in the instrument.


  1. DO NOT put any plate into the instrument after a spillage has occurred traces of fluorescent material will build up over time and impair the performance.


  1. DO report any spillages as soon as possible to the facility staff, however minor.


  1. The user is responsible for tidying up and removing all plates at the end of their experiment, within the time period of the booking.


  1. Please note that, if you are using the instrument and it is damaged or misused, the PI responsible for the user is liable for the cost of repairs.


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contacts:


Facilities Manager:

Dr. Martin Wear

Rm. Swann 3.20

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: martin.wear@ed.ac.uk


Lab Manager:

Mrs. Sandra Bruce

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: sabruce@staffmail.ed.ac.uk


Protein Technologist:

Dr. Liz Blackburn

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: E.A.Blackburn@ed.ac.uk


Protein Technologist:

Dr. Matt Nowicki

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: matthew.nowicki@ed.ac.uk



Facilities:


CTCB-Home

Protein Production Biophysical Characterisation

In Silico Screening

Training


Links:


On-line Booking

Core Column Library


Access Charges/Costs:


Access Charges/Costs

 

Location:

The University of Edinburgh,

Level 3 Michael Swann Building,

King’s Buildings,

Mayfield Rd.,

EH9 3JR,

UK


Labs 3.5 - 3.10

SpectraMax M5 & FluoroMax3