Amersham CyDye DIGE Fluor 最小标记法染料 (5 nmol)

Are there any components we should avoid in the labeling?

For minimal labeling omit all primary amines and also omit DTT until after the labeling reaction.

What to learn about the stability of the NHS-ester moiety?

The unreacted dye is considered "unstable" because of the unstable NHS-active group, and so it is stored frozen and lyophilized.

Expected shelf life 

Unreconstituted 8 - 12 months (8 months for Cy2, which, of the 3, is the most unstable)
Reconstituted in high quality DMF* 2 months (definitely true for Cy2; on the other hand, Cy3 and Cy5 are probably okay for an additional 1-2 weeks longer)
Working solution (diluted) 1 week
Stability of labeled protein (stored at -70 °C) up to 3 months (possibly longer depending on the stability of the protein itself)

*DMF is hygroscopic, picking up water from the air. Water degrades the dye's NHS-ester group. In addition, DMF breaks down to various products, including amines, which react with the NHS ester. Therefore, DMF used to reconstitute the dye should always be high-quality, and freshly opened.

Can we compare the intensity of different protein spots and relate that to their abundance?

You can to some extent but remember you are doing within-spot analysis not between-spot analysis with DIGE.

What are the general recommendations for Immunoprecipitated samples and DIGE?

General recommendations

1. The protein samples to be labeled should be in the recommended DIGE sample/lysis buffer.
2. Prior to labeling the pH of the protein sample should be at 8.5 and the following common components should not be present in high concentrations, if at all: Any primary amines (e.g. ampholytes & exogenous amines), thiols (e.g., DTT), SDS, and other untested reagents (in relation to DIGE). Ettan DIGE recommendations should be followed for all aspects of the process to ensure optimal results.

Two potential IP/DIGE options are:

1. Perform the IP, then clean up the proteins with our 2-D Clean-Up Kit, resuspend the proteins in sample/lysis buffer, quantitate the protein to ensure the correct amounts are being labeled for DIGE. Then label the proteins with the CyDye DIGE fluors as per normal for DIGE and proceed with 2-D.
2. The alternative is to label the proteins, then IP them, quantitate the resultant protein fraction to ensure enough is still present for 2-D (this may require labeling more protein than normally required depending on that particular IP method's yield), then perform 2-D as normal.

One thing that must be kept in mind is that different IP methods will potentially produce variations in the internal standard spot patterns. Consequently, this could make the results from DeCyder analysis statistically invalid so an experiment involving the comparison of samples passed through different IP methods could be problematic. The labeling of different samples and the internal standard with the different CyDye fluors, pooling them and then subjecting them to the same run through the same IP process should give good results, however, if the IP process is very variable between runs then this could again introduce variations in the internal standards increasing the variation between replicates. In summary, careful consideration must be made with regard to experimental design if IP is to be incorporated into the DIGE process.

What effect do SDS, NP-40 and lipids have on the sample preparation procedure?

Always deal with samples on a case by case basis. Labeling efficiencies can be reduced for example by the addition of 1% SDS, but 0.25% appears to have no effect. NP40 is compatible to 1%. Lipids may be a problem - but more with solubility.
Always test new samples on a 1-D gel after labeling with a single CyDye. Compare the relative intensity of the protein ladder with a known control sample which was also labeled at the same time.

How much CyDye should be added to a sample?

We recommend using 400 pmol of dye per 50 µg protein sample.

Which CyDye should be used to stain the internal standard?

If all 3 dyes are being used (for staining 2 samples plus 1 internal standard) then we recommend that Cy2 be used to stain the internal standard. It is the smallest of the 3 dyes so MW shifts away from the bulk unlabeled proteins are the smallest.

If using only 2 dyes then use Cy3 for the internal standard and Cy5 for the sample. (Cy2 is the most unstable of the dyes. Also, Cy3 and Cy 5 are most closely MW matched.)

Are there any sample types that cannot be labeled with this technology?

Not that we have found. Just ensure that the proteins are in a recommended lysis buffer (8M Urea, or 6M Urea/2M Thiourea, 4% CHAPS, 30mM Tris pH8-8.5), then there should be no problem.

How do I prepare immunoprecipitated proteins for DIGE labeling?

Immuno precipitated proteins can be removed from the beads at low pH with 100 mM glycine-HCl, pH 2.7. After neutralization with Tris base the sample can be desalted using the 2-D Cleanup kit. The sample is now ready for CyDIGE labeling using the standard protocol.

When should 2-D DIGE gels be stained with Deep Purple?

2-D DIGE gels should not be fixed or stained with Deep Purple until after all CyDye scans have been completed. Fixing before scanning for CyDyes will effect quantitation and cause a high background.

If the concentration of substances such as SDS differ in two samples, is there a risk that labeling efficiency and therefore quantification is unequally performed?

Yes-the cumulative effect of two or more compounds that have an adverse effect on labeling in one sample could affect the final quantitation.

How does the CyDye DIGE fluor staining pattern compare with the staining patterns of fluorescent post-labeled and silver stained gels?

There is little difference between the 2-D spot pattern you see with the CyDye DIGE fluors versus the pattern seen with post-labeling methods. The CyDye DIGE fluor adds approximately 500 Da to the mass of a protein. This only becomes significant towards the bottom of the gel, where you are likely to find that the unlabeled proteins have migrated slightly further than their labeled counterparts.

One protein has more lysine residues: won’t it label more than the other proteins?

This might happen, but this does not affect your quantitation. The same protein will label to the same percentage with the different CyDyes so your within-spot analysis is not affected.

What is the distribution of CyDye after the labeling reaction, i.e. how many % of 
the protein molecules will be labeled with 0, 1, 2, >2 CyDyes, respectively?

If the labeling ratio within the recommended limits such as 200 pmoles of dye to 50 ìg protein approximately 97% of protein will be unlabelled and approximately 3% labeled with one dye molecule. Dual and multiple labeled proteins are at such a low percentage that they cannot be visualized.

Can you explain the labeling ratio?

With CyDye DIGE Fluor minimal dyes 50 µg protein is labelled in each reaction. The ratio used ensures that the dyes label approximately 1-2% of lysine residues so each labelled protein carries only one dye label and is visualised as a single protein spot. The CyDye DIGE Fluor minimal dyes therefore only label a small proportion of the total protein in a sample. For that reason, this type of labelling has been called minimal labelling.

How does the sensitivity of Ettan DIGE compare with post-electrophoresis staining methods?

Ettan DIGE has the same sensitivity range as silver staining.

Can you use DNase and RNase in the samples?

Yes but these may show up on the gel depending on the molecular weight and type of gel.

What is the effect of nucleic acids in the sample?

High nucleic acid content can cause gel smearing in the second dimension, but this can be reduced by sufficient sonication to shear the DNA.

What is the recommended sample load of DIGE Dye?

It is recommended that 50 µg of each labeled protein sample is combined for each 1st dimension gel (i.e., IPG strip).

What protein concentration range is recommended prior to start of labeling?

Protein samples at higher concentrations such as 10mg/ml label more efficiently than dilute solutions such as less than 2mg/ml.
Sample preparation should not exceed 10mg/ml. Otherwise, loss of protein and horizontal or vertical streaking due to protein aggregation and precipitation may occur (Gorg et al 2000 "The current state of two-dimensional electrophoresis with immobilised pH gradients" Electrophoresis 21. 1037-1053.)

Why does an increase in temperature of the sample result in lowered fluorescence?

Fluorescence is just one mechanism by which a molecule can return to its ground state after it has been excited by absorption of radiation. Another mechanism is radiationless transition, in which energy is carried away by collisions with other molecules rather than by re-emission of light. Raising the temperature increases the number of collisions between molecules, and thus, increases the chance that molecules return to the ground state WITHOUT fluorescence emission of radiation.

In DIGE applications, can ordinary CyDye fluors be used instead of CyDye DIGE fluors?

No, only CyDye DIGE fluors are size and charge-matched to bind without altering protein pI and molecular weight.

Is it possible that differences in post-translational modifications between samples will cause differences in labeling efficiency?

If there are post-translational modifications that affect the labeling it will affect the labeling with the different CyDyes to the same degree.

Is it possible to make quantitative comparison of two samples in which the total protein concentration is different?

Yes, but the statistical significance of your differences will be affected. We always recommend that you label and load an equal amount of the different samples on the same gel.

What method do you recommend for protein concentration determinations?

Use a detergent or thiourea compatible protein assay kit, e.g. Protein Determination Reagent or Ettan 2D Quant Kit.

Can I store the gels and image them later?

Imaging should be done immediately to avoid loss of resolution due to diffusion of the spots. Unless doing Sypro Ruby post-staining, the gels are not fixed (because fixing can affect the relative quantification of the Cy2-, Cy3- and Cy-5-labeled samples).

Why do all the DIGE protocols stipulate the use of Pharmalyte 3-10 rather than IPG Buffer?

Customers can use Pharmalytes or IPG buffers; it is up to the customer to test which is better for their results because one may work better than the other on their particular sample. We have just happened to use Pharmalytes for most of our experiments and this is reflected in our recommended protocols.

What samples have been labeled using the Ettan DIGE technology?

A. Mammalian cell lines, mammalian tissues, mammalian biopsy, bacteria, yeast, plant, Drosophila, and biological fluids such as plasma and serum.