• What type of samples can be analyzed?

Most biological and clinical samples can be analyzed, including blood, serum, plasma, tissue, cultured cells, tumor biopsies, laser micro dissected samples. Plant, nematode, microbial, insect and fish samples can also be analyzed.  

• What type of buffer is suitable/best for 2D-DIGE experiments?

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 www.itsibio.com reduces processing time and ensures reproducibility and quality results. 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 losses and changes in the protein content.    

• What are the steps for the 2D-DIGE process at ITSI-Biosciences?
• - Samples are precipitated to remove interfering substances (if necessary).   
• - The samples are then re-suspended in 2D-DIGE buffer.
• - Protein concentration is determined using the ToPA Bradford protein assay.
• - If necessary, protein samples are qualified using the Agilent BioAnalyzer.
• - 50ug of each protein is labeled with 200pmole Cy3 or Cy5 dye respectively, for the minimal labeling approach. If sample concentration is limiting, 30ug 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 internal standard.
• - The labeled samples are loaded on a 24cm IEF strip, pH 3-10. IEF strips with different 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 gels are scanned on a Typhoon Digital Imager at 3 different wavelengths.
• - The images are analyzed using DeCyder analysis software (GE Healthcare) and statistical analysis is performed.
• - Spots representing proteins-of-interest picked and tryptic digested with spot handling robots and identified by MALDI-TOF MS or 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.

What is the amount of protein required to run a 2D-DIGE gel?
The minimum amount of protein required for a 2D-DIGE gel is 100µg for each sample.

• What is the difference between 2D-Gel and 2D-DIGE?

In the DIGE (Difference in-gel electrophoresis) process, protein samples are directly labeled with fluorescent dyes 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-gel 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.

• What is the turnaround time for 2D-DIGE analysis?

The turnaround time for 2D-DIGE analysis is 4-5 business days. This will be dependent on the number of samples submitted.

•  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 samples not used in the experiment will a) be returned to the customer at the end of the experiment if requested (a fee may be charged for the postage), b) immediately destroyed after the experiment if directed by the customer or c) stored for up to 5 years with no charge to the customer if space is available, before discarding.

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 varies and depends  on the type and the number of samples submitted for analysis. To request a quote please contact technical support at 1-814-262-7331 or itsi@itsibio.com.

 

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