Amersham CyDye DIGE Fluor Labeling Kit for Scarce Samples
CyDye DIGE Fluor minimal dyes and saturation dyes are size- and charge-matched fluorescent dyes specifically designed for detecting protein abundance differences in 2-D Fluorescence Difference Gel Electrophoresis using Ettan DIGE system.
FAQ
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 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 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.)
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.)
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.
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.
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.
How does the sensitivity of Ettan DIGE compare with post-electrophoresis staining methods?
Ettan DIGE has the same sensitivity range as silver staining.
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.
How much CyDye should be added to a sample?
We recommend using 400 pmol of dye per 50 µg protein sample.
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.
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.
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.
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.
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).
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.
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.
What are the general recommendations for Immunoprecipitated samples and DIGE?
General recommendations
- The protein samples to be labeled should be in the recommended DIGE sample/lysis buffer.
- 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:
- 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.
- 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Select symptom:
| Possible cause | Suggested remedy |
|---|---|
The CyDye DIGE Fluor saturation dyes exceeded their expiry date prior to reconstitution, resulting in poor protein labeling. |
Check the expiry date on the dye pack label. |
Primary amines (e.g. pharmalytes or ampholytes) or thiols (e.g. dithiothreitol DTT) are present in the labeling reaction competing with the protein for dye. |
Omit all exogenous primary amines and thiols from the labeling reaction. |
Interfering components are present in the labeling reaction at too high a concentration, resulting in poor protein labeling. |
Remove the compounds from the labeling reaction if not essential. If they are essential test if the reduction in labeling efficiency can be counterbalanced by increasing TCEP/dye concentration. Investigate this using the method described in instructions CyDye DIGE Fluor Labeling Kit for Scarce Samples. |
There is little or no protein in the protein lysate, or less lysate was loaded on the gel. |
Test this by checking the protein lysate concentration using Protein Determination Assay. |
The protein lysate concentration is too low i.e. less than 0.55 mg/ml. |
Make a new batch of protein lysate reducing the volume of cell lysis buffer to increase the protein concentration. Alternatively, precipitate the proteins and resuspend them in a smaller volume of cell lysis buffer. |
The reconstituted dye has been stored for too long, resulting in poor protein labeling. After reconstitution CyDye DIGE Fluor saturation dyes are only stable and useable until the expiry date detailed on the tube, or for 8 weeks, whichever is sooner. |
Check the expiry date on the dye pack label and do not use dye that has been reconstituted for 8 weeks or more |
The dimetylformamide (DMF) used to reconstitute CyDye DIGE Fluor saturation dyes, was of poor quality or has been opened for longer than 3 months, resulting in poor protein labeling. |
Always use 99.8% anhydrous DMF to reconstitute CyDye DIGE Fluor saturation dyes. Breakdown products of DMF include amines that compete with the protein for dye during the labeling step or cause dye degradation. |
The dyes have been exposed to light for long periods of time, resulting in loss of fluorescent signal. |
Always store CyDye DIGE Fluor saturation dyes, in the dark. |
The dyes have been left out of the freezer (-15°C to -30°C) for a long period of time, resulting in poor protein labeling. |
Always store CyDye DIGE Fluor saturation dyes at |
The wrong focal plane has been set on the Typhoon Variable Mode Imager. |
Set the focal plane to “+3 mm” for gels assembled between standard glass plates or “platen” for gels placed directly on the platen. |
The Typhoon Variable Mode Imager settings are inappropriate. |
Ensure all parameters comply with recommended instrument settings. |
The pH of the protein lysate is less than pH 7.8, resulting in poor protein labeling. This may be due to cell lysis causing a drop in pH or incomplete removal of the cell wash buffer prior to addition of the cell lysis buffer. |
Ensure the Tris buffer is present at 30 mM. Increase the pH of the cell lysis buffer by the addition of a small volume of 50 mM NaOH or cell lysis buffer at pH 9.5. |
The pH of the protein lysate is more than pH 8.2, resulting in poor protein labeling. |
Decrease the pH of the cell lysis buffer by the addition of a small volume of 50 mM HCl. |
| Possible cause | Suggested remedy |
|---|---|
The amount of tris carboxyethyl phosphine (TCEP)/dye used is too low. |
Refer back to gels from the labeling optimization experiment to determine the correct amount. Repeat labeling optimization if necessary. |
There is insufficient sodium dodecyl sulfate (SDS) in the running buffer. |
Prepare a fresh batch of running buffer ensuring that it contains 0.2% SDS. |
The concentration of Pharmalytes is too high. Pharmalytes complex with proteins at their isoelectric point. If too much Pharmalyte is present it may be difficult for proteins to resolubilize for transfer into the second dimension. |
Ensure that the recommended concentration of Pharmalytes is used (no greater than 1% during rehydration). |
| Possible cause | Suggested remedy |
|---|---|
The amount of tris carboxyethyl phosphine (TCEP)/dye used is too low. |
Refer back to gels from the labeling optimization experiment to determine the correct amount. Repeat labeling optimization if necessary. |
The sample press option has not been selected when scanning. |
Ensure that the sample press option is used with assembled gels. |
Poor mixing during labeling, causing non-uniform labeling. |
Mix vigorously by pipetting following each reagent´addition during the labeling protocol. |
| Possible cause | Suggested remedy |
|---|---|
The amount of tris carboxyethyl phosphine (TCEP)/dye used is too high. |
Refer back to gels from the labeling optimization experiment to determine the correct amount. Repeat labeling optimization if necessary |
The pH of the protein lysate is above pH 8.2, resulting in non-specific labeling of lysine residues. |
Check the pH of the cell lysis buffer and reduce if necessary by adding a small volume of 50 mM HCl. |
| Possible cause | Suggested remedy |
|---|---|
The dimethylformamide (DMF) used to reconstitute CyDye was of poor quality or it has been longer than 3 months since it was first opened. |
To reconstitute CyDye always use >99.8% anhydrous DMF from a fresh bottle, or from a bottle that is less than 3 months from the day it was first opened and purged with argon gas before recapping. Even if recapped, once exposed to air DMF breaks down to amines which react with the CyDye NHS ester. In addition, DMF is hygroscopic and water in DMF can hydrolyze the NHS ester moiety. To be safe, discard bottles of DMF once opened. If you have no access to argon gas then this is imperative. |
CyDye that has been diluted to working concentration is too old. |
Once diluted to working concentration, the CyDye is only usable for a maximum of 2 weeks. |
The protein lysate concentration is too low (i.e. less than 1 mg/ml). |
a. Check the concentration using a protein assay that is not affected by detergent or thiourea, such as PlusOne 2-D Quant Kit. |
Thiol agents (e.g., DTT over 2 mg/ml) or other substances such as SDS (over 0.2%) may be present in the labeling reaction at too high a concentration. |
a. Dilute the protein lysate with lysis buffer containing no DTT. |
Primary amines such as Pharmalyte or other carrier ampholytes are present in the labeling reaction competing with the protein for CyDye. Dye was thus free to react with substrates other than lysine residues and so labeling efficiency was poor. |
a. Dilute the protein lysate with lysis buffer containing no amines. |
No lysine was present. |
DIGE requires lysine for labeling. |
Labeling efficiency was too poor as the binding reaction was maintained at pH 8. |
a. The lysis of the cells has caused a drop in the pH. Increase the buffering capacity of the Lysis buffer to 40 mM Tris (50 mM is the recommended maximum for Tris). |
An incorrect CyDye to protein ratio was used. |
We recommend using 400 pmol of CyDye DIGE fluor per 50 µg of protein. If there is also present a large concentration of other components which can react with the NHS ester moiety of the CyDye then more CyDye can be used (up to 2 nmol per 50 µg of protein). Note that in this regard it is important that the entire contents of the vial be mixed thoroughly with the added DMF, to ensure that proper amounts of CyDye are delivered to the labeling reaction. |
CyDye has been exposed to higher temperature than -20 °C for a long period of time. |
Prevented action: Always store CyDye at -15 °C to -30 °C and only remove them for short periods to remove a small aliquot. |
CyDye has expired, or been exposed to light for long periods of time and has thus photodegraded. |
Prevented action: Always store CyDye in the dark. |
| Possible cause | Suggested remedy |
|---|---|
Proteins not denatured or solubilized as much as possible. |
Use a combination of chaotropes in the lysis buffer, such as 7 M urea / 2 M thiourea. |
| Possible cause | Suggested remedy |
|---|---|
The lysis of the cells has caused a drop in the pH. |
Increase the buffering capacity of the Lysis buffer to 40 mM Tris (50 mM is the recommended maximum for Tris). |
The cell wash buffer was not completely removed prior to addition of the lysis buffer. |
Increase the pH of the lysis buffer by the addition of a small volume of 50 mM NaOH. Or add an equal volume of the lysis buffer that is at pH 9.5. |
| Possible cause | Suggested remedy |
|---|---|
Contaminant proteins have been introduced into the sample prior to the labeling reaction. |
Check that gloves are used throughout the procedure. |
| Possible cause | Suggested remedy |
|---|---|
- |
As with all 2-D techniques, the separation of proteins in both first and second dimensions is important. If there is evidence that spots are too close together to be confidently separated by the DeCyder software then it is advisable to run a more appropriate 1st dimension pH range or 2nd dimension gel (e.g. use narrow pH range or a gradient gel respectively). |
| Possible cause | Suggested remedy |
|---|---|
Ordinary CyDye fluors were used instead of CyDye DIGE fluors. |
Only CyDye DIGE fluors are size- and charge-matched to bind without altering protein pI. |