Frequently asked questions
For additional information, visit the Protein Analysis Blog.
Questions about Protein Identification Analysis
Polyacrylamide gel is the most commonly applied type. The protein separation can occur one-dimensionally in SDS-PAGE, which separates proteins by mass weight, or two-dimensionally in 2D-PAGE, where proteins are first separated by an isoelectric point and then by mass weight. To identify the separated proteins, you can combine all standard gel types for protein separation with in-gel trypsin digestion and MS-analysis.
Coomassie blue is a non-covalent dye used for post-electrophoretic staining. All different types of Coomassie blue work for protein identification analysis. Silver staining, with silver nitrate or silver diamine, has a higher sensitivity than Coomassie blue, but the chemicals used might affect the MS-based protein analysis in some protocols. Optimized silver staining method for MS analysis can be found in our protocols (at the Support menu). More recent fluorescent dyes, such as Sypro Ruby and Deep Purple, are also compatible with MS protein identification.
Clear Coomassie or Silver stained bands for protein identification analysis provide good data. E.g., with 50 femtomoles or more loaded onto the protein gel. Look into our Protein Analysis Blog for more information.
Use a new clean scalpel blade for cutting out the desired protein band/spot. Cut the gel on a clean glass plate while wearing gloves at all times. Place the gel piece in a new Eppendorf tube. It is now ready for protein identification analysis.
Keratin is a naturally occurring protein found in, e.g., hair, dead skin, latex gloves, DTT, buffers, and wool clothing. Minimizing keratin contamination by wearing a lab coat and non-latex gloves is essential. Additionally, everything in touch with the gel should be free of dust, e.g., washed with 70% ethanol. Perform the in-gel digestion in a clean work area like a laminar flow hood. You can find additional information in our Protein Analysis Blog.
– Proteolytic digestion that generates peptides.
– Extraction of the peptides from gel-pieces if in-gel digestion
– Peptide cleanup and concentration on small scale HPLC material
– MS and MS/MS analysis by MALDI mass spectrometry or ESI-LC mass spectrometry.
– Protein amino acid sequence database search by software such as Mascot (MatrixScience)
The sequence coverage expresses the number of amino acids in a protein sequence found in the peptides observed by mass spectrometry.
Alphalyse provides several post-translational modification services, including disulfide bridge analysis and glycosylation analysis. Besides that, we also provide the determination of physicochemical and structural properties.
We can identify several proteins in a gel band excised from 1D SDS PAGE by trypsin digestion followed by nanoLC-MS/MS peptide analysis and database searching.
To identify proteins observed in a Western blot, see our Protein Analysis Blog.
We can identify multiple proteins in your gel band with trypsin digestion and subsequent nano LC-MS/MS peptide analysis. We search against the UniProt database with all known proteins.
This issue can be due to several problems. Perhaps the proteins in your sample are not present in the UniProt database, e.g., if the protein is entirely novel and the organism genome is not sequenced. Look into our Protein Analysis Blog for more information about this situation.