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Integrated Structural Biology Grenoble
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Presentation MALLS

Presentation

MALS measurements work by calculating the amount of scattered light at each angle detected. The intensity of light scattering of a solution is directly proportional to the average molecular weight and to the concentration of its components.
Our Multi-Angle Laser Light Scattering (MALLS), UV Absorbance and Refractive Index (RI) coupled to a size-exclusion chromatography system allows the simultaneous determination of the molecular weight of each component of a sample.

For more information about theoretical aspect please visit Wyatt’s website

 SEC–MALS separation of BSA and haemoglobin (Wyatt)
SEC–MALS separation of BSA and haemoglobin (Wyatt)

Example MALLS Figure

Application

  • Determination of concentration and molecular weight
  • Determination of aggregation and oligomerisation states, homogeneity of the sample
  • Determination of conformational changes :
    • Protein-protein interactions
    • Protein-nucelotides interactions
  • It is a method often appropriated for studying interactions of membrane protein and/or glycosylated proteins

Key words

Size-exclusion chromatography, Multi-Angle Laser Light Scattering, Protein concentration, Oligomerisation states, stoichiometry of complexes

Staff

Platform engineer : Caroline Mas
Professor Marc Jamin
Office : CIBB room 020
Platform : CIBB room 001

Specific Equipment

Wyatt Dawn Heleos II - Multi-Angle static Light Scattering
Wyatt Optilab T-rex – Refractometer
Hitachi Elite LaChrom UV detector L-2400
Hitachi Elite LaChrom Pump L-2130

Access mode

Users must read the conditions of use of the instruments as well as of the management of the services provided :

General Rules- Biophysics Plateform
General Rules- Biophysics Plateform
Management of the services provided
SEC-MALLS Guidelines

Instrument allocation after user training

  • It is mandatory to be trained by the platform manger before using this instrument. The first two days of use will be done with the assistance of the manager.
  • After demonstrating his autonomy, the user will be able to use the instrument in complete independence
  • The platform is accessible to local PSB researchers, external academics (Instruct) and industrials
  • Service : routine experiments performed for punctual users
  • Scientific collaboration : projects in which the Biophysical platform is strongly involved. In this case, experiments are designed, performed and analyzed in tandem between the scientific responsible and the applicant.
  • You have to bring your own Size exclusion chromatography column
  • The platform manager will provide assistance for :
    • _ experimental design
    • _ data collection
    • _ samples considerations
    • _ data analysis
  • Training on the instrument is provided the day of your first experiment
  • Contact the platform manage to arrange a meeting if necessary

Cost

Academics : Participation to the maintenance fees
Industrials : Please contact us for cost information

Location

The Biophysical platform is located in the Carl-Ivar Brändén Building, ground floor, room 001 (Shared Building C).

Samples

Only non-pathogenic biological samples are accepted. Decree of the July 18, 1994 establishing the list of biological pathogens, amended by Decrees of April 17, 1997 and June 30, 1998 (Decrees in French). The list of biological pathogens is available on the website of the IPBS.

Guidelines

• The equilibration of the system/column is done overnight. Usually, the column is connected at the end of the afternoon the day before the experiment and equilibrated overnight at 0.2 mL/min.

• Precaution for the use of the columns
­ Flow used is 0.5 mL/min
­ Column to be connected on the MALLS system has to be in water or buffer
­ Elution buffer should be de-gazed and filtered at 0.22µm
­ The platform DO NOT run columns upside down & DO NOT accept packed columns.

• The biophysical platform do not provide SEC columns, user have to bring their own columns FERSHLY CLEANED. Please note that the column listed below are accepted :
­ Superdex 200 30/100
­ Superdex 75 30/100
­ Superose 6 30/100
­ If another column has to be used, please contact the Platform Manager before.

• Note that :
­ the last injection of the day should not be done after 4:00 pm in order to allow time to set up the equilibration of the next user.
­ 1 run takes 50 min at 0.5 mL/min ( max 5 samples per day)
­ one buffer and one column per day
­ Book number of days accordingly

• Instructions for the samples
­ The sample volume required for one injection is 55 µl
­ The purity of the sample should be 90%
­ The sample concentration should be between 2 and 10 mg/mL
­ Samples have to be centrifuged and/or filtered before injection
­ Samples are not stored
­ A guideline concentration is 2 mg/ml of protein and a minimum volume of 50 µl is required. Lower concentrations may not give very good light scattering signal (sample dependant). Small proteins (< 15 kDa) will give low light scattering signals and may need a higher concentration.

• For each experiment, data acquisition record must be completed on the SEC-MALLS logbook :

  • Record the details of the experiments
    o Name / Surname
    o Date
    o Column
    o Buffer
    o Light Scattering (LS) Value (should be 0,03)
    o List of experiments collected :
    o ExpXXXX BSA 4 mg/mL 50uL
  • Record any anomalies

• Standard protocol for using the SEC-MALLS system and reference manuals for the software ASTRA can be consulted on site.

• At the end of each booking period, you must put the system in filtered/de-gazed H2O, Ensure that the experience stops

How to make a request

To make a booking request, submit the sample form and fill the request form.

SEC-MALLS booking

Biophisics Platform Booking Calendar

Cost

Academics : Participation to the maintenance fees
Industrials : Please contact us for cost information

Follow up / acknowledgements

The user agrees to promote the obtained results by mentioning the platform in the acknowledgements in case of publication or scientific communication and to communicate the reference of the article to the responsible for the platform.

Service / Providing access mode : Acknowledgements in publications and oral presentations

Collaboration : Co-authors in publications and oral presentations.

Acknowledgements in publications :

Please find below the sentence that has to be written in all publications based on our UMS platforms :
"This work used the platforms of the Grenoble Instruct-ERIC center (ISBG ; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). We thank Caroline Mas for assistance and/or access to the Biophysical platform."