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Gel Electrophoresis

The Two-Dimensional Electrophoresis capabilities include 7cm mini gel, 13cm 18cm and 24cm large format gel systems. We routinely run a wide variety of complex protein mixtures on these various formats, with the mini-gel systems generally used to obtain an initial overview of a sample's protein distribution. The large gel systems are then used to analyse and investigate regions of interest with the aim of visualising detailed sections of the proteome. Proteins profiles are visualised using either coomassie or mass spectrometry compatible silver stains.

Theory

  • 2D gels offer outstanding resolving power for complex samples and current in gel digestion techniques and MALDI based PMF makes it an essential proteomics tool.
  • The use of 1D gels should not be overlooked as it is useful for the analysis of samples not suited to 2D gels such as membrane proteins
  • This method is used for the separation and identification of proteins in a complex mixture using two separate dimensions that are run perpendicular to one another.
  • This allows a complex biological sample to be separated over a larger area, increasing the resolution of each component.

The 1st Dimension

  • The 1st dimension uses isoelectric focusing (IEF) which separates proteins based on their overall charge or the pI which is a property of a protein due to the amino-acid composition.
  • IEF uses pH gradients that can be established by adding ampholytes or immobilines to an acrylamide gel. Ampholytes are a mixture of different amphoteric molecules that have a range of isoelectric point values.
  • Immobilized strips are dehydrated which allows rehydration of the strips directly with the sample to be separated.

The 2nd Dimension

  • The 2nd dimension separates proteins based their molecular weight.
  • A number of gel gradients (polyacrylamide SDS-PAGE) concentrations and different buffering systems can be used in the second dimension.
  • This method is essentially the same as a 1D SDS-PAGE gel, proteins separated by mass based on a near equivalent charge to mass ration due to the presence of the SDS detergent anion.

Example 2D gel

Example 2D gel

Typical Experiments and Procedures

  • The investigation of global protein expression of an organism or a specific tissue which allows for rapid protein identification using mass spectrometry to generate peptide fingerprint data.
  • Differential expression is a technique that compares two or more samples to study differences in their protein expression in a differential system. An example could be that a diseased and healthy tissue are differentially examined to elucidate proteins responsible for the pathology of disease, or the effects of a drug on treated versus untreated tissue samples.

Sample Information and Preparation Required

  • Protein samples should be provided as concentrated as possible and details of concentration and buffer type should be given. If these are unknown then for and extra charge the sample can be cleaned up and quantitated.
  • Amount of Material: The following table provides a guideline to the amounts of protein required for various gel types:
    - mini gel 7cm 10-50 µg total protein
    - large gel 24cm 100-500 µg total protein
  • Sample information: Any useful details relating to the potential behaviour of your proteins of interest in a PAGE system such as the nature of the proteins eg membrane or soluble.
  • Sample buffer: samples should be supplied free of the following components which interfere with the first dimension isoelectric focusing step: salt >50mM, nucleic acids, polysaccharides, lipids, SDS and other ionic detergents. A desalting procedure should be carried out prior to sample submission or if necessary we can perform an appropriate clean up.
  • Approximate amount of protein: An estimated concentration of protein in ug/mL is essential. The recommended lower limit is 350 ug/mL. If unknown we can include a quantitation in the procedure.
  • IPG Strip pH range: If the pI of the target proteins are known then a narrow pI range may be chosen to maximize resolution otherwise, a broad range (3-10) strip will be used.
  • SDS-PAGE gel percentage: An appropriate gel must be chosen for obtaining an optimal separation of your proteins of interest depending on the estimated molecular weight range.
  • An extensive sample preparation guide can be found at the following website:
    Expert Protein Analysis System (external site)
  • Protein pre-fractionation: In some cases such as human serum a single to a few proteins may be present in a sample in such high abundance that they interfere with proper electrophoresis or inhibit resolution of other proteins.
  • Proteases: Protease activity can result in poor quality gels. Their activity varies between different organisms and tissues. To limit their activity, the sample should be kept on ice at all times during sample preparation or a protease inhibitor cocktail can be added.
  • Sample Solubilisation: The preparation of sample for 2D electrophoresis generally requires the use of a buffer that solubilizes and denatures proteins such as 8M urea. This separates the proteins into individual species and improves the separation by IEF.
  • Centrifugation: All samples should be centrifuged before IEF to sediment particulates present that can cause smearing and blockage of the polyacrylamide gel matrix.
  • Protein Estimation is an important step to know the concentration of proteins in a sample before it can be loaded onto the IEF gel to avoid over loading or under loading the IPG strip.
  • Urea a chaotrope used to solubilise proteins disrupts hydrogen and hydrophobic bonds, and has no effect on the native charge of a protein. In solution, at higher temperatures, urea forms an equilibrium with isocyanate which can cause carbamylation of primary amino groups such as the N-terminus or lysine or arginine sample proteins and thus modifies the protein charge resulting in streaks of spots across the gel caused by titration of the charge of a particular protein.
  • Staining.
    - Coomassie Blue staining is a quick, easy and cheap and multiple protocols and pre-made reagents are available. This stain is not specific and will also detect non protein components.
    - Silver staining can give excellent results but is a time consuming and expensive technique that can interfere with MS analysis if not done properly. The advantage of silver staining is its sensitivity. MS friendly silver staining protocols are available.
    - An alternative to silver stain is Sypro Ruby has the same level of sensitivity and is MS compatible however is more expensive.