Comparative and Semi-Quantitative Proteomics Using 2D Gel Electrophoresis (2D-DIGE) Technology

ITSIBIO » Comparative and Semi-Quantitative Proteomics Using 2D 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.

2D-DIGE Analysis
1SP1Sample Preparation 1Homogenization, protein quantitation using ToPA Bradford protein assay kit.Per sample
2SP2Sample Preparation 2Includes, precipitation, buffer exchange, protein quantitation, etc. and the use of ToPI-DIGE, ToPREP and ToPA kits.Per sample
3SP3Sample Preparation 3Depletion of abundant protein e.g. bovine or human serum albumin using ITSI ASK-Column kit.Per sample
4SP4Sample Preparation 4Depletion of abundant protein e.g. bovine or human serum albumin using ITSI ASK-Solvent kit.Per sample
5SP5Sample Preparation 5Depletion IgY Column Depletion of 14 abundant proteins from serum /plasma using IgY column.Per sample
6SP9Sample Preparation 9Urine protein isolation and concentration. Service includes Isolation, partial purification, and concentration of urine proteins using ITSI Urine Protein Isolation kit.Per sample
7S302D-DIGETwo- 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
8S40PRobotic spot pickingSelection and picking of fluorescent labels protein spots from 2D-DIGE gels using fully automated ETTAN spot picking robot.1-96 spots
9S40DRobotic spot digestionIn gel digestion of protein spots picked from 2D-DIGE gels using a fully automated ETTAN spot digestion robot.1-96 spots
10S50.60LC60-MS/MSPeptide 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