Why Does GFP Show Two Bands on SDS-PAGE After FPLC, and How Can It Be Purified More Precisely?
When green fluorescent protein (GFP) exhibits two distinct bands on SDS-PAGE after purification via fast protein liquid chromatography (FPLC), several factors may account for this phenomenon:
1. Partial Protein Degradation
Proteolytic cleavage during expression, extraction, or storage may generate protein fragments with lower molecular weights than the intact GFP, resulting in additional bands.
2. Post-Translational Modifications
Modifications such as phosphorylation or glycosylation can alter the apparent molecular weight of GFP, leading to a secondary band.
3. Isoforms
Different conformational states or isoforms of GFP may migrate differently during SDS-PAGE.
4. Co-Purified Impurities
Despite FPLC purification, proteins of similar molecular weight to GFP may co-elute and appear as additional bands.
To achieve higher purity, the following strategies may be implemented:
1. Optimization of FPLC Conditions
Depending on the column type employed (e.g., gel filtration, ion exchange, or affinity chromatography), adjustments to the elution gradient or buffer composition may improve resolution and separation efficiency.
2. Sequential Chromatographic Purification
A two-step purification scheme, such as initial affinity chromatography for primary enrichment, followed by gel filtration or ion exchange chromatography, can enhance the removal of contaminants.
3. Improved Sample Handling and Extraction Protocols
Incorporating adequate concentrations of protease inhibitors during lysis and minimizing processing time can mitigate degradation of GFP.
4. Thermal Treatment
Exploiting differences in thermal stability between GFP and contaminating proteins, brief incubation at an empirically determined temperature followed by cooling and centrifugation may selectively remove heat-labile impurities.
5. Control Experiments Using Alternative Expression Systems
Expressing GFP in a different host can help determine whether the appearance of two bands is host-dependent.
6. Proteolytic Mapping
Partial digestion of GFP with specific proteases, followed by SDS-PAGE analysis, can clarify whether the two bands correspond to distinct protein species or related fragments.
By applying these approaches, it is possible to enhance the purity of GFP preparations and elucidate the underlying causes of the dual-band pattern observed in SDS-PAGE analysis.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
