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.
|ELISA||Standard 2D Western Blot||2D DIGE Western Blot|
|Number of gels for each Ab||Two||One|
|Detection Method||Silver Staining||CyDye Labeling|
|Detect HCP Composition|
|Detect protein modification|
|Detect protein degradation|
|Protein and WB in same gel|
|In-gel protein and WB comparison|
|Inaccurate: In-direct reaction of antibodies with the antigen’s associated proteins||Inaccurate: Protein spots counted from the gel, NOT from membrane||Accurate: Protein spots counted directly from the membrane|
|False negative:||Error caused by:||Accurate:|
|Consistency & Reproducibility||Lower||Lower||Higher|
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.