SEC-MALS

 
 

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Instrument:

Viscotek MALS-20 and VE3580 RI detector attached to an ÅKTA 10 Purifier with micro-flow components.






















Please click here for individual platform/resource costings.

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What is SEC-MALS?

light passing through solvent interacts with the molecules in it, leading to scattering off the axis of the incident light beam (660 nm). Measurement of the scattered lights intensity is detected at different angles to the beam (in this case a Viscotek MALS-20 system). If an additional solute of different refractive index is present this leads to additional excess scattering dependent not only on it’s concentration but also on its molar mass (for large particles, typically with diameter ~ 1/20th of the wavelength of the incident light, there may be an angular dependency on the scattering intensity). In a suitably calibrated system this may be used to determine the molar mass of the solute. The angular dependence for large particles (>400 - 500 kDa) may be used to estimate size (i.e. radius of gyration).

Accurate determination of the solute concentration is vital for determination of molar masses. The system also includes a refractive index detector (a VE3580 RI instrument). Proteins have a very consistent concentration dependent effect on refractive index of solutions. Attaching these two instruments down stream of an HPLC system and size exclusion column, provides a very accurate method of determining the molar mass of partitioned samples.

The MALS system will provide accurate determination of molar mass for homogeneous samples. It will also indicate where peaks are composed of mixtures of different oligomers (mass changes across the peak) or conformers (where the peak shape changes or indicates indicates multiple species but mass is the same). The software provides estimates of polydispersity. For larger proteins (>400 - 500 kDa) the scattered light intensity varies with angle of detection in a way that depends on the size (strictly radius of gyration) of the molecule. If the molar mass is also determined then inferences can be drawn about the shape of the molecule - globular or rod shaped etc.


The system can be connected to a fraction collector if necessary (100 µl minimum volume).


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Columns:


The EPPF has the following columns for exclusive use on the SEC-MALS system.


Superdex Peptide 10/300 GL column - dynamic range for proteins; 100 Da. - 5 kDa.


Superdex 75 Increase 10/300 GL column - dynamic range for proteins; 1 kDa. - 70 kDa.


Superdex 200 Increase 10/300 GL column - dynamic range for proteins; 10 kDa. - 600 kDa.


Superose-6 Increase 10/300 GL column - dynamic range for proteins; 5 kDa. - 5 MDa.


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Samples and buffers:


A guideline concentration is 1 mg/ml protein in the sample and a minimum  volume of 100 µl is required.  Lower concentrations may give very good signal to noise but this is very sample dependant. Remember that  smller proteins (< 15 kDa) give low light scattering signals and may need a higher concentration.

All precipitates and insoluble material must be removed by centrifugation or pre-filtration before running your sample.

Users should provide their own buffers unless arranged previously with EPPF staff. Buffers must be made from analytical grade (or better) reagents in Grade-1 water and filtered through 0.22 µm filters and degassed. Small particles in the eluent or shed from the column will cause high backgrounds in the light scattering signal thus degrading the data. Matching the sample buffer with the sun buffer is advised if possible. This will minimise the refractive index signal seen at the column inclusion volume.

When arranging time on the system please remember to allow for column equilibration and cleaning steps - the equilibration is conveniently done overnight and recommended.

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Location:

The University of Edinburgh,

Level 3 Michael Swann Building,

King’s Buildings,

Mayfield Rd.,

EH9 3JR,

UK


Lab 3.16

SEC-MALS - Viscotek MALS20

contacts:


Facilities Manager:

Dr. Martin Wear

Rm. Swann 3.20

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: martin.wear@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