Quality control by electron microscopy

Introduction

The EM quality control service by negative staining can:

• verify the homogeneity of a sample (aggregated or not).
• determine the degree of oligomerization of a protein.
• allow image analysis and 3D reconstruction of protein complexes (collaboration).

Keywords

Electron microscopy, negative staining, homogeneity

Dedicated staff

• Daphna Fenel
• Christine Moriscot
• Guy Schoehn

Equipment

Tecnai12 120 kV LaB6 with an Orius 1000 GATAN camera

• Calibration of the microscope: twice a year
• Calibration of the camera: once a week

Accessibility

The platform is accessible to members of the PSB, academia, and industry.

How do I apply?

By writing to: ibs-plateforme-em.contact ibs.fr
You will receive a reply with a proposed date.
Please consider an usual delay of 2 weeks between your request and the proposed date.

Sample requirements

The minimum size of molecules that can be observed is 100 kDa. Detergent and glycerol are best avoided in the buffer.
The minimum quantity required is 30 microliters of the sample at about 0.2 mg/ml and 100 microliters of the buffer for dilution in case necessary. After the experiment, the samples will be either taken back by the customer, or they will be stored for 2 weeks before elimation.
The samples must be pure.
Samples must have security level L1 (or L2 if fixed) and conform to the corresponding GMO statement.

Procedure

After preparation of the grid by negative staining (mica flotation technique or stain-on-grid technique), five different areas on the grid are inspected. A minimum of 10 images of the sample are taken at magnification varying from 10000x to 60000x depending on the sample (except for special cases).

Results

After the microscope session, a report containing the most representative images of the sample together with comments approved by the team manager is sent (via ftp CEA or the IBS intranet) within three weeks after the receipt of the samples (except in the case of instrument breakdown). The rest of the images are available upon request.

Data storage

The data are stored for 3 years.

Costs

see Prices

Acknowledgment

If you use these results (from the service platform or collaboration) please add:

• In your acknowledgments:
  « This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UAR 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-0005-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA.»
• In your publications:
  Fenel, Daphna1,2,3; Moriscot, Christine1,2,3; Schoehn, Guy1,2,3

1CEA, Institut de Biologie Structurale Jean-Pierre Ebel, UMR5075 Grenoble France.
2CNRS, Institut de Biologie Structurale Jean-Pierre Ebel, UMR5075 Grenoble France.
3Université Grenoble Alpes, Institut de Biologie Structurale Jean-Pierre Ebel, UMR5075 Grenoble France.

Location

IBS, ground floor, electron microscopy and methods group.

Quality

The platform is certified ISO9001-NFX 50-900