Comparative and semi-quantitative proteomics using Two-Dimensional Difference In-Gel Electrophoresis (2D-DIGE) Technology.

ITSI-Biosciences utilizes validated sample preparation kits/protocols, robotic spot pickers/digesters, DeCyder difference-in-gel analysis software and advanced mass spectrometers to offer an integrated 2D-DIGE – MS/MS proteomics solution. Proteomic analysis usually starts from sample preparation, to analyses by mass spectrometry (LC-MS/MS) and protein identification and annotation. Data obtained after 2D-DIGE analysis may include protein identity, pI, molecular weight and post translational modifications.

ITSI-Biosciences has provided 2D-DIGE analysis and proteomics services to NIH, industry, biotechnology companies, pharmaceutical companies, Research Institutes and Universities. Our more than 15 years of experience with 2D-DIGE analysis, proteomics, mass spectrometry and protein expression profiling enables us to offer reproducible picture perfect gels at a competitive price and guaranteed faster turnaround time. We have also developed a variety of complementary 2D-DIGE kits to streamline and standardize protein isolation, protein quantitation, protein qualification and the 2D-DIGE analysis step, thereby reducing the overall time and cost of performing 2D-DIGE analysis.

2D-DIGE offers all the advantages of the classical 2D-PAGE, and most importantly overcomes the inherent disadvantage of gel-to-gel variation and reproducibility problem inherent with the traditional 2D-PAGE approach. 2D-DIGE is presently the most versatile gel-based approach for comparative and semi-quantitative proteomics. Distinct fluorescent tags e.g. Cy 3, 5 and 2 are used to label protein samples and a universal internal standard prior to 1st/2nd dimension electrophoresis. An automated software program is used to detect, quantify and annotate differentially expressed proteins. 2D-DIGE analysis can be applied to protein expression profiling and comparative proteomics by using DeCyder software to identify proteins with statistically-significant difference in expression. This provides unique opportunities for researches in fields including cancer, neuroscience, vision, cardiovascular, Alzheimer, Parkinson, etc, to find important proteins associated with the condition. 2D-DIGE – LC/MS/MS analysis can also be applied to identify post translationally modified proteins (PTM) that are important in cell signaling and protein trafficking.

1 SP1 Sample Preparation 1 Homogenization, protein quantitation using ToPA Bradford protein assay kit. Per sample
2 SP2 Sample Preparation 2 Includes, precipitation, buffer exchange, protein quantitation, etc. and the use of ToPI-DIGE, ToPREP and ToPA kits. Per sample
3 SP3 Sample Preparation 3 Depletion of abundant protein e.g. bovine or human serum albumin using ITSI ASK-Column kit. Per sample
4 SP4 Sample Preparation 4 Depletion of abundant protein e.g. bovine or human serum albumin using ITSI ASK-Solvent kit. Per sample
5 SP5 Sample Preparation 5 Depletion IgY Column Depletion of 14 abundant proteins from serum /plasma using IgY column. Per sample
6 SP9 Sample Preparation 9 Urine protein isolation and concentration. Service includes Isolation, partial purification, and concentration of urine proteins using ITSI Urine Protein Isolation kit. Per sample
7 S30 2D-DIGE

Two- Dimensional Difference in-Gel Electrophoresis (2D- DIGE). Quantitative and comparative proteomics using 2D-DIGE. Services include:


  • Experimental design,
  • Sample preparation to isolate total proteins using ToPI- DIGE Protein Isolation Kit,
  • Fractionation, quantitation and qualification of proteins,
  • 2D-DIGE analysis using Cy2, Cy3 and Cy5, scanning of gels using a DIGE- enabled digital scanner and
  • Biological variation analysis of gel images to identify differentially expressed proteins with statistically significant difference.
2 – 10 Samples
8 S40P Robotic spot picking Selection and picking of fluorescent labels protein spots from 2D-DIGE gels using fully automated ETTAN spot picking robot. 1-96 spots
9 S40D Robotic spot digestion In gel digestion of protein spots picked from 2D-DIGE gels using a fully automated ETTAN spot digestion robot. 1-96 spots
10 S50.60 LC60-MS/MS

Peptide sequencing and protein identification by liquid chromatography – tandem mass spectrometry of pure protein samples. Services include:


  • Tandem mass spectrometry analysis using nano-LC/MS/MS,
  • Database search and
  • Identified protein annotation – 60 min run.
Per sample