TWO DIMENSIONAL DIFFERENCE IN GEL ELECTROPHORESIS (2D-DIGE)
What type of samples can be analyzed?
Any sample from which proteins can be isolated can be analyzed. This includes all biological and clinical samples can be analyzed, such as blood, serum, plasma, tissue, cultured cells, culture media, tumor biopsies, and laser micro dissected samples. All animal, plant, nematode, microbial, insect and fish samples can also be analyzed.
What type of buffer is suitable/best for 2D-DIGE experiments?
Basically, any buffer system suitable for the upstream process can be accommodated. However, it may be necessary to include a precipitation and buffer exchange step. If samples are submitted in a different buffer then a buffer exchange step will be required prior to the 2D-DIGE process. This step may lead to unavoidable losses and changes in the protein content.
The 2D-DIGE process is very sensitive and the use of inappropriate buffer systems could affect the labeling efficiency, reproducibility and accuracy of the experiment. The best buffer for 2D-DIGE is the buffer system that is specifically prepared for 2D-DIGE. Based on more than 8 years experience we recommend the ToPI-DIGE (Total Protein Isolation Kit for 2D Difference Gel Electrophoresis) specifically formulated and validated for 2D-DIGE. This cost-effective buffer system available at https://www.itsibio.com/topi_dige.html reduces processing time and ensures reproducibility and quality results.
What are the steps for the 2D-DIGE process at ITSI-Biosciences?
– Proteins are isolated from samples using ToPI-DIGE kit.
– If client sent samples already partially processed, then samples are precipitated to remove interfering substances (if necessary) and the samples are re-suspended in 2D-DIGE compatible buffer.
– Protein concentration is determined using the ToPA Bradford protein assay.
– If necessary, protein samples are qualified using the Agilent BioAnalyzer.
– 50µg of each protein is labeled with 200pmole Cy3 or Cy5 dye respectively, for the minimal labeling approach. If sample concentration is limiting, 30µg protein can be analyzed using the saturated labeling approach. Cy2 is used to label equal amounts of protein from all samples and used as the universal internal standard/control.
– The labeled samples are loaded on a 24cm IEF strip, pH 3-10. IEF strips with different (narrow and wide) pH ranges can be also used.
– The strips are rehydrated in the presence of the samples for 12 hrs at 30 volts.
– The rehydrated IEF strips are focused for a total of 65,000 volt hours.
– The focused strips are loaded onto a 24cm x 20cm, 12.5% SDS-PAGE gel, and run for 4 hours. The gel strength can be varied to select for low or high molecular weight proteins.
– The gels are scanned on a DIGE-enabled Typhoon Digital Imager at 3 different wavelengths.
– The images are analyzed using DeCyder analysis software (GE Healthcare). Depending on the experimental design, the statistical analysis is performed with the Difference In gel Analysis (DIA) module or the Biological Variation Analysis (BVA) module of DeCyder. The data obtained will be in p-values and ANOVA.
– Spots representing proteins-of-interest picked and tryptically digested with spot handling robots and identified by LC/MS/MS.
What is the maximum number of samples that can be run on a 2D-DIGE gel?
We can run up to three samples on one 2D-DIGE. Two samples will be labeled with Cy3 and Cy5 and the third sample labeled with Cy2 is the universal internal control that allows gel-to-gel comparison.
What is the amount of protein required to run a 2D-DIGE gel?
The minimum amount of an individual total protein sample recommended for a 2D-DIGE experiment is 30µg. However, we recommend sending at least 100µg of each sample. If protein amount is limiting then the “saturated” labeling approach is used. If protein concentration is not limiting then the “minimal” labeling approach is used. We have experience analyzing less than 30µg (as low as 5µg has been analyzed) but then only the high abundant proteins will be detected and downstream identification of a candidate spot by mass spectrometry becomes challenging due to the small amount of peptides that will be available for sequencing.
What is the difference between 2D-DIGE and Classical 2D(2D-PAGE)?
In the DIGE (Difference in-gel electrophoresis) process, protein samples are directly labeled with fluorescent dyes (typically Cy2, Cy3 and Cy5) before being separated by 2D electrophoresis. Since samples can be labeled with different fluorescent dyes they can be multiplexed on one gel, reducing gel to gel variations. In a 2D-PAGE only one sample per gel can be analyzed. Also to visualize a 2D- gel the gel must be stained, increasing the background and reducing sensitivity of detection, whereas in a 2D-DIGE gel the labeled proteins can be detected without staining the gel allowing low abundant proteins to be detected. With 2D-DIGE a few number of gels can be used for comparative proteomics.
What is the turnaround time for 2D-DIGE analysis?
The turnaround time for the 2D-DIGE aspect is 3 – 5 days for 10 samples or less. The turnaround time for an entire project that includes spot picking, in gel digestion and identification of the candidate proteins of interest by mass spectrometry is 2 – 3 weeks for most experiments.
Who owns the results generated?
All data generated are owned by the customer.
What happens to samples that are not completely used in the experiments?
Any sample not used in the experiment will a) be returned to the customer at the end of the experiment if requested (a fee will be charged for the shipping), b) immediately destroyed after the experiment, if directed by the customer or c) stored for up to 6 months with no charge to the customer if no instruction was received and space is available. At the end of 6 months the samples are discarded to make space or de-identified. Discarded or de-identified samples cannot be returned to customers.
Does ITSI-Biosciences have a confidentiality policy regarding samples and results?
Yes. All experiments and transactions are confidential. ITSI-Biosciences will not use the results even for advertisement unless permission is given in writing by the owner. Every employee signs a legally binding non-disclosure and confidentiality agreement at the start of employment at ITSI-Biosciences.
What is the cost per sample for 2D-DIGE analysis?
ITSI-Biosciences price for 2D-DIGE process is very competitive and there are often special or seasonal promotions from the Dye manufacturer that can be passed on to customers. Since the cost will vary as a function of the experiment, we encourage clients to download and complete the Project Summary/Sample Submission Form available here to enable us better estimate the project.
Please contact us at: 1-814-262-7331 or firstname.lastname@example.org if you have any more question.