What Should Be Done When SDS-PAGE Bands Contain Multiple Protein Bands in Protein Sequencing?
When a section of an SDS gel contains more than one protein band, these bands may correspond to distinct proteins or various isoforms, including alternative translation products or forms modified post-translationally. To accurately identify the amino acid sequence of each protein individually, it is necessary to further separate and purify these proteins. The following methods are typically employed to address SDS gels with multiple protein bands:
1. SDS-PAGE Optimization
Initially, perform a secondary electrophoresis on the SDS gel segment with multiple bands, optimizing electrophoresis conditions to achieve enhanced separation of the diverse protein bands.
2. Band Excision and Purification
Following separation, each protein band can be individually excised and purified using advanced techniques such as fragment excision, gel slicing, or specialized protein purification methods.
3. Mass Spectrometry Sequencing
The purified protein bands can be subjected to mass spectrometry sequencing techniques, including MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) or LC-MS/MS (Liquid Chromatography coupled with Tandem Mass Spectrometry), to obtain detailed amino acid sequence information.
4. Database Correlation
Finally, the mass spectrometry data can be matched against protein databases to identify protein information that corresponds with the experimental results.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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