2D DIGE Western Blot vs Standard 2D WB / ELISA / AAE

HCP Antibody coverage can be performed by standard 2D Western Blot, ELISA, and AAE. Applied Biomics has developed a 2D DIGE Western Blot, an advanced technique that overcomes the drawbacks in all the platforms above:

  • High consistency: Perfect alignment of HCP and WB images from the same gel, eliminating any gel-to-gel or gel-to-membrane variations as seen in the standard 2D WBs.
  • Cost-effective: Since only one gel is needed, it saves the cost of running duplicate gels.
  • High accuracy: Fluorescent CyDye labeling allows accurate quantitation of protein and WB spots.
  • High sensitivity: Fluorescent CyDye labeling has a higher sensitivity and wider dynamic range than silver staining.

ELISAStandard 2D Western Blot2D DIGE Western Blot
Number of gels for each AbTwoOne
Detection MethodSilver StainingCyDye Labeling
Detect HCP CompositionImageImageImage
Detect protein modificationImageImageImage
Detect protein degradationImageImageImage
Protein and WB in same gelImageImageImage
In-gel protein and WB comparisonImageImageImage
AccuracyLowLowHigh
Inaccurate: In-direct reaction of antibodies with the antigen’s associated proteinsInaccurate: Protein spots counted from the gel, NOT from membraneAccurate: Protein spots counted directly from the membrane
False negative:
  • Coated Ab does not capture all HCPs
  • Surface denaturation of coating Ab can affect detection
Error caused by:
  • Gel to gel variation
  • Gel to membrane variation
Accurate:
  • Protein and WesternBlot image on the same gel, avoiding any ambiguity
Consistency & ReproducibilityLowerLowerHigher

AAE, based on antibody immuno-binding with antigen under the native condition, has limitations in quantitating antibody coverage:

  • False positive: a) direct and indirect associate proteins; b) antigens present in the HCP antibody; c) co-purified serum proteins
  • False negative: a) associate proteins as a big complex will block antibody binding; b) some binding HCPs or their degraded fragments cannot be eluted
  • Not all HCP proteins/fragments can be affinity purified, and the HCP profile is totally changed after AAE
  • In addition, AAE still need be coupled with downstream LCMS or 2D Western blot, the lengthy process introduces more variations and inconsistency.