How to determine the content of dimers under conditions with and without drug treatment?
To accurately compare dimer content under conditions with and without drug treatment, it is essential to clarify: protein properties (solubility, tendency to aggregate), detection environment (in vitro purified protein or cell/tissue lysate), and whether the goal is qualitative or quantitative analysis. The following strategies can be considered:
1. Gel Electrophoresis Combined with Quantification
1. Principle:Dimers relative to monomers can maintain conformational differences under non-reducing, low-denaturation conditions.
2. Method:
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Use non-reducing SDS-PAGE or Native-PAGE for separation;
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After staining, use image analysis software (e.g., ImageJ) to perform grayscale integration of the monomer and dimer bands to calculate the dimer ratio;
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Ensure the sample loading is within the linear range to avoid aggregation or overloading that leads to false positives.
3. Applicability:Simple and intuitive, but may underestimate weakly bound or dynamically balanced dimers.
2. SEC (Size-Exclusion Chromatography)/ HPLC
1. Principle:Separation by molecular weight, allowing distinction between monomer and dimer peaks.
2. Method:
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Quantify peak area using a standard curve (external standard proteins or known concentration of purified target protein);
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It is recommended to combine with MALS (Multi-Angle Light Scattering) or UV detection to improve accuracy.
3. Applicability:Can provide a more accurate ratio and detect drug-induced dimerization changes.
3. Crosslinking Combined with Mass Spectrometry (Crosslinking-MS) or Chemical Crosslinking + Western
1. Principle:Drugs may alter dimerization dynamics, and crosslinking can capture specific dimers.
2. Method:
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Use mild crosslinkers (e.g., DSS, BS3) to stabilize interactions;
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Combine with SDS-PAGE or mass spectrometry to analyze the proportion of crosslinked products.
3. Applicability:Suitable for transient or low-affinity dimers, but crosslinking conditions need to be strictly optimized.
4. Biophysical Methods
1. MALS (Multi-Angle Light Scattering):Can directly provide molecular weight distribution in solution;
2. AUC (Analytical Ultracentrifugation):Measures sedimentation coefficients to distinguish between monomers and dimers;
3. Applicability:Accurate but requires specialized instruments and purified samples.
Suggested Workflow
1. First confirm the sample state (whether original binding state needs to be maintained, whether the drug is stably present).
2. Use SEC-MALS or Native-PAGE quantification as the first choice; if conditions are limited, combine non-reducing SDS-PAGE + densitometry for estimation.
3. It is recommended to set up biological replicates in parallel and maintain equal total protein and the same buffer system under both conditions.
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