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HCP Analysis: Advantages of 2D DIGE Western Blot Over ELISA & Standard 2D WB

 

 

 

  ELISA Standard 2D Western Blot 2D DIGE Western Blot


Number of gels for each Ab

  Two One


Detection Method

  Silver Staining CyDye Labeling


HCP Composition and profiling

No


Detect protein modification

No


Detect protein degradation

No


Protein and WB in same gel

No No


In-gel protein and WB comparison

No No


Accuracy

Low Low High

Inaccurate: In-direct reaction of antibodies with the antigen’s associated proteins

Protein spots are counted from the gel, NOT from the membrane

Transferred protein spots are scanned directly from the membrane

False negative: 1) Coated Ab does not capture all HCP; 2) Surface denaturation of coating Ab can affect detection

Error caused by: 1) Gel to gel variation; 2) Gel to membrane variation

Accurate: Protein and Antibody signal on the same gel, thus avoiding any ambiguity


Consistency and Reproducibility

Lower Lower Higher