Membrane Proteins

Membrane protein characterisation - without purification
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What does FIDA bring to Membrane Protein research?

01

Save sample material

  • Down to 40 nl of sample
  • No purification needed – high tolerance toward impurities
  • No restriction on buffer composition
  • Delivers data needed to optimised membrane protein expression conditions and increase yield
  • Possible sample recovery
02

Tagged or label-free

  • Tag flexibility: GFPs, HIS, Alpha tag, FLAG and similar
  • Possibility of label-free characterisation of purified membrane protein
03

8 quality parameters in 1 run

  • Assess structural changes
  • Validate structures against reference models using built-in PDB Correlator (integration with Protein Data Bank)
  • Know the absolute size (Rh) measurement
  • Determine your sample’s Polydispersity Index (PDI)
  • Check for oligomeric state
  • Quantify aggregation behaviour of your sample
  • Detect ligand-binding and quantify Kd
04

Short assay optimisation and validation

  • Save time with dedicated Assay Design tool
  • First principle methods - allow you to avoid assumptions
  • Built-in QC accelerates your optimisation process
  • Absolute readouts and structural correlation facilitate efficient and reliable data validation
05

Temperature controlled – retain structure and function

  • Keep your sample stored at 5°C while operating in walk-away mode
  • Independent temperature control in sample and capillary compartment

Efficient analysis and
rapid decision-making

With the ability to utilize sample volumes as low as 4μL (typically 10μL) for the analyte and 40 nL for the indicator, as well as the flexibility to operate directly in unpurified sample matrices, FIDA users can obtain a greater number of data points in a significantly reduced timeframe.

The users place unpurified samples in the autosampler’s 96 well plates or 2x50 vials, and after a few hours they are able to make an informed choice on which membrane protein to purify for further analysis.

Fast and easy – the FIDA way.

Down to
4μL
Analyte
40nL
Indicator Use

Detergent screening for solubilisation – 85% faster

Save sample material

  • 10 to 100 times less sample volume in comparison to other technologies
  • 0.12 uL per detergent (for triplicates)
  • No restrictions on buffer compositions

Increased throughput

  • Less than 3h of experiment time for 12 detergents
  • Analysis time reduced by 85% (compared to FSEC)
  • Walk-away automation
  • Screening of up to two 96 well plates

Work with any detergent in any concentration

  • No clogging
  • 50 nM detection sensitivity level (for GFP)

Upfront quality control – in a single run

  • Automatic QC ahead of in-depth analysis
  • Quantitative assessment of aggregation
  • Binding site functionality
  • Polydispersity index allows users to assess uniformity in solubilisation
  • Upfront detection improves user efficiency by eliminating suboptimal detergents

How does FIDA
accelerate detergent screening
for protein solubilisation?

FIDA users are able to shorten their experiment time by 85%, saving days’ worth of time. How? Thanks to FIDA’s walk-away autosampler and the ability to identify issues with stability, oligomeric states and binding site functionality early in the workflow.

1.5 ul

singlets

<3h

Triplicates

Sample Volume

12 Detergents

Experimental Time

12 Detergents

Benefits

Keeps the cell lysate at 5°C while operating in walk-away mode
Quantitative detection of activation and binding in complex membrane constructs
Lower sample consumption
Speed to data
In-depth quality control
Informed troubleshooting

Features

Triple temperature control
Autosampler
Quantification of aggregates
Quantification of solubilised membrane protein
Polydispersity index
Oligomerisation

Measures

Accurate determination of dilute phase concentration
Relative droplet size distributions
Kinetics of droplet formation and maturation into amyloid fibrils
Determination of binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds

Targeting common Membrane Protein Research Challenges

Challenge
Expression and purification
FIDA users can determine the in-solution concentration of membrane proteins. This allows them to optimise protein crystallisation conditions and to monitor protein expression and purification processes.

With just 0.12 uL per detergent (for triplicates) in detergent screening and down to 40nl of unpurified sample needed for characterisation, FIDA uses 10 to 100 less sample volume compared to other available technologies.
Stability and conformational heterogeneity.
Triple temperature control and possibility to work in unpurified matrices allows FIDA users to increase their level of control over membrane protein stability.
Functional characterisation
Working in crude matrices allows FIDA users to increase the applicability of their results. Additionally, the polydispersity index ensures that sample heterogeneity is considered in functional characterisation.
Structural characterisation
The size measurement delivered by FIDA allows our users to monitor changes as a result of interactions with other proteins or ligands. This information is then used to analyse the structure and function of membrane proteins of interest.

FIDA is currently used in cryo-EM sample preparation processes, as well as in nanobody clone selection. Our users can apply a single technology to characterise their membrane proteins, screen detergents, produce nanobodies to immobilise their membrane proteins and run cryo-EM sample preparation.
Effective drug development
FIDA measures the change in the size of the membrane protein complex when a ligand is bound, making it an optimal tool for evaluating protein-ligand interactions. This information allows FIDA users to identify potential ligands for drug discovery.

Do you need to cryo-scan your membrane proteins?

Read about the use of FIDA in CRYO-EM
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