CyDye DIGE Fluors CyDye DIGE Fluor minimal dyes CyDye DIGE Fluor Labelling Kit for Scarce Samples CyDye DIGE Fluor minimal dyes ..... CyDye DIGE Fluor minimal dyes are exceptional for multicolor analysis, offering bright and intense colors with narrow excitation and emission bands. They are spectrally distinct, making them ideal for multicolor detection. CyDye DIGE Fluor minimal dyes utilize these benefits but are also specially developed to be size and charge matched specifically for Ettan DIGE System. The protein samples and the internal standard are each labelled with a different CyDye DIGE Fluor minimal dye. These labelled samples are then combined, run on an isoelectric focusing (IEF) gel in the first dimension, and separated by SDS-PAGE in the second dimension. Electrophoresis is simplified with IPGphor™ System or Multiphor™ System with Immobiline™ DryStrip gels in the first dimension and the Ettan DALT electrophoresis system in the second dimension. The protein samples and the internal standard are each labelled with a different CyDye DIGE Fluor minimal dye. These labelled samples are then combined, run on an isoelectric focusing (IEF) gel in the first dimension, and separated by SDS-PAGE in the second dimension. Electrophoresis is simplified with IPGphor™ System or Multiphor™ System with Immobiline™ DryStrip gels in the first dimension and the Ettan DALT electrophoresis system in the second dimension. The ability to multiplex different CyDye DIGE Fluor minimal dye labelled samples on the same gel means that the different samples will be subject to exactly the same 1st and 2nd dimension running conditions. Consequently the same protein labeled with any of the CyDye DIGE Fluor minimal dyes and separated on the same gel will migrate to the same position on the 2–D gel and overlay. This limits experimental variation and ensures accurate within-gel matching. One lysine group labelled per protein CyDye DIGE Fluor minimal dyes have an NHS-ester reactive group, and are designed to covalently attach to the epsilon amino group of lysine of proteins via an amide linkage. The ratio of dye to protein has been specifically designed to ensure the dyes are limiting in the reaction. As a result the dyes label approximately 1-2% of the available proteins and then only on a single lysine per protein i.e. one dye per protein, or "minimal labelling". The lysine amino acid in proteins carries an intrinsic single positive charge at neutral or acidic pH. CyDye DIGE Fluor minimal dyes also carry a single positive charge which, when coupled to the lysine, replaces the lysine's single positive charge with its own, ensuring that the pI of the labelled protein does not significantly alter compared to the same unlabelled protein. CyDye DIGE Fluors: the facts Size and charge matched — labelled samples co-migrate with the gel Bright and highly sensitive — enable the minimal labelling technique to be used Photostable — minimal loss of signal during labelling, separation and scanning pH insensitive — no change in signal over wide pH range used during first dimension (IEF) separation Spectrally distinct — discrete signal from each fluor contributes to high accuracy with no cross-talk Experimental Design Minimal Dyes An example of a recommended experimental set-up designed to derive statistical data on differences between control and two treatment regimes A and B are outlined below. For the control and two treatment regimes, four biological replicates are included (1-4). The internal standard (a pool of equal amounts from all samples: four controls and eight treated) is labelled with CyDye DIGE Fluor Cy2 minimal dye and run on every 2-D gel. Each control and treated sample is labelled with either CyDye DIGE Fluor Cy3 or Cy5 minimal dye and loaded on gels as indicated below. Gel Cy2 Cy3 Cy5 1 Pooled internal standard Control 1 Sample A1 2 Pooled internal standard Sample B2 Control 2 3 Pooled internal standard Control 3 Sample B1 4 Pooled internal standard Sample B3 Sample A3 5 Pooled internal standard Sample B4 Sample A4 6 Pooled internal standard Sample A2 Control 4

Documentation CyDye DIGE fluor Data File CyDye DIGE Fluors (minimal dyes) for Ettan DIGE Protocol Booklet . CyDye DIGE Fluor Labelling Kit for Scarce Samples CyDye™ DIGE Fluor saturation dyes Two new CyDye DIGE Fluor dyes are available from Amersham Biosciences for multiplexing in a 2-D gel. Two kits, one for analytical work only and the other for analytical work and the preparation of a preparative gel, contain Cy3 and Cy5 saturation dyes. These kits will enable a complete analysis of precious samples that are available in scarce amounts. The recommended amount to label in this kit will be 5ug. Like CyDye DIGE Fluor minimal dyes for general sample labelling the CyDye DIGE Fluor saturation dyes are size and charge matched, spectrally distinct, bright and intense in colour with narrow excitation and emission bands. Two dyes are available in each kit Cy3 and Cy5, one kit contains an additional vial of Cy3 to label a preparative gel. CyDye™ DIGE Fluor Labelling Kit for Scarce Samples Two color overlaid image of Mouse brain labelled with 5mg of CyDye DIGE Fluor Cy3 and Cy5 saturation dye from CyDye DIGE Fluor Labelling Kit for Scarce Samples. The protein samples and the internal standard (used on every gel) are each labeled with a different CyDye DIGE Fluor saturation dye. These labeled samples are then combined, run on an isoelectric focusing (IEF) gel in the first dimension, and separated by SDS-PAGE in the second dimension. Electrophoresis is simplified with IPGphor™ System or Multiphor™ System with Immobiline™ DryStrip gels in the first dimension and the Ettan DALT electrophoresis system in the second dimension. The ability to multiplex different CyDye DIGE Fluor labelled samples on the same gel means that the different samples will be subject to exactly the same 1st and 2nd dimension running conditions. Consequently the same protein labelled with the two CyDye DIGE Fluor saturation dyes and separated on the same gel will migrate to the same position on the 2–D gel and overlay. This limits experimental variation and ensures accurate within-gel matching. Labelling of all available cysteine groups in a protein CyDye DIGE Fluor dyes from the Scarce Sample Labelling Kit have a maleimide reactive group, and are designed to covalently bond to the thiol group of cysteine residues of proteins via a thioether linkage. To achieve maximal labelling of cysteine residues the protocol uses a high fluor to protein labelling ratio. This type of labelling method labels all available cysteines on each protein under the conditions used resulting in the majority of cysteine groups in a protein from a sample, being labelled. For this reason, this method has been called saturation labelling. The CyDye DIGE Fluor saturation dyes have a net neutral charge and therefore do not alter the charge on a protein after labelling. They are also matched for molecular weight ensuring that the same protein from a standard and test sample labelled with the different dyes will overlay when separated on a single gel. CyDye DIGE Fluors: the facts Size and charge matched — labelled samples co-migrate with the gel Bright and highly sensitive — enable the labelling of 5µgs of sample Photostable — minimal loss of signal during labelling, separation and scanning pH insensitive — no change in signal over wide pH range used during first dimension (IEF) separation Spectrally distinct — discrete signal from each fluor contributes to high accuracy with no cross-talk Experimental Design Saturation Dyes An example of a recommended experimental set-up designed to derive statistical data on differences between control and two treatment regimes A and B are outlined below. For the control and two treatment regimes, four biological replicates are included (1-4). The internal standard (a pool of equal amounts from all samples: four controls and eight treated) is labelled with CyDye DIGE Fluor Cy3 saturation dye and run on every 2-D gel. Each control and treated sample is labelled with either CyDye DIGE Fluor Cy5 saturation dye and loaded on gels as indicated below. Gel Cy3 Cy5 1 Pooled internal standard Control 1 2 Pooled internal standard Control 2 3 Pooled internal standard Control 3 4 Pooled internal standard Control 4 5 Pooled internal standard Sample A1 6 Pooled internal standard Sample A2 7 Pooled internal standard Sample A3 8 Pooled internal standard Sample A4 9 Pooled internal standard Sample B1 10 Pooled internal standard Sample B2 11 Pooled internal standard Sample B3 12 Pooled internal standard Sample B4

Documentation

CyDye DIGE Fluor Labelling Kit for Scarce Samples Instruction Booklet

CyDye DIGE Fluor Labelling Kit for Scarce Samples Protocol reminder card