Frequently asked questions
For additional information, have a look at our Protein Analysis Blog.
Questions about Protein Identification Analysis
The most commonly applied type is the polyacrylamide gel. The protein separation can occur within one dimension, as in SDS-PAGE where proteins are separated by mass, or in two dimensions, as in 2D-PAGE where proteins are separated by isoelectric point in the first dimension and by mass in the second. All standard gel types for protein separation can be used in combination with in-gel trypsin digestion and MS-analysis to identify the separated proteins.
Coomassie blue is a non-covalent dye used for post-electrophoretic staining, all different types of Coomassie blue can be used 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 dyes are fluorescent such as Sypro Ruby and Deep Purple that are also compatible with MS protein identification.
For protein identification analysis really good data are obtained with clear Coomassie or Silver stained bands, e.g. with 50 femtomole or more loaded onto the protein gel. Look into our Protein Analysis Blog for more information.
Cut the gel on a clean glass plate, while wearing gloves at all times. Use a new clean scalpel blade for cutting out the desired protein band/spot. Place the gel piece in a clean 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. It is important to minimize keratin contamination by wearing a lab coat and non-latex gloves. Additionally, everything in touch with the gel should be free of dust, e.g. by being washed with 70% ethanol. The in-gel digestion must be performed in a clean work area like a laminar flow hood. Additional information is found in our Protein Analysis Blog.
You can perform de-novo sequencing of monoclonal antibodies using tandem MS (nanoLC-MS/MS) and database searching with PEAKS and Mascot software. The method is especially useful if the parent hybridoma cell is not available for DNA sequencing but can also be utilized when the amino acid sequence does not match the DNA sequence. After the analysis, N-terminal Edman sequencing can be performed to obtain the heavy and light chain of the monoclonal antibody.
– 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.
– Database searching in protein amino acid sequence databases by software such as Mascot (MatrixScience)
The number of amino acids in a protein sequence that were found in the peptides observed by mass spectrometry.
We offer several services that determines post translational protein modifications, including disulfide bridge analysis and glycosylation analysis. Other than that, we also offer determination of physiochemical 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.
For identification of proteins observed in a Western blot see our Protein Analysis Blog.
With trypsin digestion and subsequent nano LC-MS/MS peptide analysis, we can identify multiple proteins in your gel band. We search against the UniProt database with all known proteins.
This issue can be due to several problems. It is possible that the proteins in your sample are not present in the UniProt database if the protein is completely novel and the organism genome not sequenced. Look into our Protein Analysis Blog for more information about this situation.
We offer de-novo peptide sequencing to obtain the full protein sequence but cannot identify the protein if it is completely novel. The sequencing is performed by nanoLC-MS/MS and database searching with PEAKS and Mascot software. We can also provide you with the high-quality spectra to enable you to investigate the peptides more when the organism has had its genome sequenced, as mentioned in our Protein Analysis Blog. Furthermore, N-terminal Edman degradation can be utilized to determine the first 40 amino acids of the protein’s N-terminal sequence.