Analysis of Proteins by Two-dimensional Electrophoresis (2-DE/2D-PAGE) (CAT#: STEM-ET-0344-ZJF)

Introduction

Two-dimensional gel electrophoresis (2-DE) is the benchmark method for the separation of intact proteins from complex mixtures. Many hundred different protein spots can be displayed from complex samples. Based on 2-DE, we provide proteomic service including but not limited to the following: • Protein cataloguing • Protein quantification • Analysis of protein species and isoform distribution • Analysis of posttranslational modifications • Protein and antibody quality control • Host cell protein analysis • Western blot We accept samples including but not limited to the following: • Purified proteins • Frozen cell pellets or tissues • Serum (with/without immunodepletion of abundant proteins) and plasma • Body fluids (e.g. urine, cerebrospinal fluid (CSF), saliva, bronchoalveolar lavage) • Tissues (e.g. myocardium, brain, kidney, liver, colon, artery tissue) • Blood cells (e.g. monocytes, lymphocytes) • Cell cultures (e.g. HeLa cells, human embryonic stem cells) • Fermentation cultures (e.g. E. coli, bacillus caldolyticus, cyanobacteria) • Microorganisms (e.g. trypanosoma brucei, bdellovibrio bacteriovorus, helicobacter pylorii, saccharomyces spp, penicillium spp) • Plants (e.g. omphalotus olearius, arabidopsis thaliana, taxus baccata, potato tuber, tomato fruits) • Other (e.g. insects, mollusca) If you have any requirements or questions, please contact us for more details.

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Principle Applications Procedure Materials Notes Related Products

Principle

Two-dimensional gel electrophoresis (2DE) is a form of gel electrophoresis commonly used to separate and analyze proteins by molecular charge and molecular size. Proteins are first solubilised in a denaturing buffer containing a neutral chaotrope, a zwitterionic or neutral detergent, and a reducing agent. In the first dimension, proteins are separated by their isoelectric point (pl) in isoelectric focusing (IEF). In the second dimension, proteins are separated by molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Each protein is therefore resolved at a unique isoelectric point/molecular weight coordinate. After visualisation by staining proteome changes are revealed by gel image analysis, and protein spots of interest excised and identified by mass spectrometry sequence analysis combined with database comparison.

Applications

Molecular biology, proteomics, genetics, biomedical, clinical, pharmaceutical, forensic, industrial, and food analysis

Procedure

1. Preparation: Proteins are usually solubilised in a denaturing buffer containing a neutral chaotrope, a zwitterionic or neutral detergent, and a reducing agent. The procedures of sample preparation are dependent on the sample type.
2. Isoelectric focusing (IEF): In a particular pH gradient, proteins are separated by differences in their isoelectric points.
3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): Proteins are separated based on the differences in their molecular weight.
4. Visualisation: Visualization of proteins by silver or coomassie brilliant blue staining.
5. Determination: Determination of molecular weight, pl, amount and others results by gel image analysis and mass spectrometry sequence analysis combined with database comparison.

Materials

• Isoelectric focusing and gel electrophoresis apparatus
• Sample solution
• Buffer solution and SDS-polyacrylamide gel

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