Why Does Purified Protein Show 60kDa Instead of the Expected 107kDa During SDS-PAGE
If a purified protein displays a significantly lower apparent molecular weight on SDS-PAGE than expected (e.g., expected 107 kDa but observed as 60 kDa), several potential explanations should be considered:
1. Protein Degradation
The most common explanation is partial degradation of the protein during extraction or purification, often caused by endogenous protease activity. To prevent this, protease inhibitors should be added during protein extraction and purification, and all procedures should be carried out as rapidly as possible.
2. Incorrect Start Codon Selection
In heterologous expression systems, an improperly selected start codon in the expression construct may lead to translation initiation from an unintended site, resulting in a truncated protein product.
3. RNA Degradation or Incomplete Transcription
In cases where the protein is expressed from transcribed RNA, degradation of the RNA or incomplete transcription may result in a shortened or aberrant translation product.
4. Post-Translational Modifications
Certain post-translational modifications can increase protein susceptibility to degradation or cause anomalous migration behavior on SDS-PAGE.
5. Unusual Protein Structure
In rare cases, secondary, tertiary, or quaternary structural features may affect electrophoretic mobility in SDS-PAGE. However, such structural effects typically do not account for discrepancies as large as observed here.
6. Technical errors
Experimental artifacts, such as errors in sample preparation or protein quantification, may also result in incorrect estimation of molecular weight.
Solutions:
1. Mass Spectrometry Analysis
Characterize the protein sample using mass spectrometry to determine its amino acid sequence and verify whether it matches the expected sequence.
2. Western Blot
Perform Western blot analysis using specific antibodies to further confirm the identity of the protein.
3. Redesign and Validation of Expression Construct
Ensure the expression vector is properly designed, and confirm its sequence through DNA sequencing.
4. Optimization of Purification Conditions
Improve purification procedures by including protease inhibitors, maintaining low temperatures, and minimizing processing time.
5. RNA Quality Assessment
If the protein is transcribed from RNA, evaluate the integrity and quality of the RNA to rule out degradation or incomplete transcription.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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