2D Western Blot Service
We offer the most comprehensive 2D Western Blot (WB) analysis by using fluorescent labeling and high-resolution, large-format 2D gel. Our report provides Protein, WB and Protein/WB overlay images, allowing the direct visualization of antibody detection that no other platform can achieve. The fluorescent labeling also allows accurate quantitation of WB signals. This platform has been successfully applied in the following areas:
- Identify protein spots interacted with antibody
- Study HCP antibody coverage
- Study different post-translational modifications (PTMs)
The unique feature of our 2D DIGE Western Blot allows 3 study designs (note #2 and 3 are not feasible in standard WB).
- Study design 1: Sample-1, Western Blot
- Study design 2: Sample-1, Sample-2, Western Blot – Western Blot reflects the combined effects of both samples
- Study design 3: Sample-1, Western Blot-1, Western Blot-2 (this 2-color WB is feasible if the two primary antibody are generated in different animal species)
Our 2D Western Blot platform offers significant advantages over standard 2D Western blot:
| Standard 2D Western Blot | 2D DIGE Western Blot | |
|---|---|---|
| Number of gels for each Ab | Two | One |
| Detection Method | Silver Staining * | CyDye Labeling * |
| Protein and WB in same gel |  |  |
| In-gel protein and WB comparison | | |
| Accuracy | Low | High |
| Protein spots are counted from the gel, NOT from the membrane | Transferred protein spots are scanned directly from the membrane | |
| 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 | Higher |
| * Sensitivity of fluorescent CyDye labeling is higher than that of silver staining (view details) | ||
2D Western Blot experimental procedure
Tissues or cells
Protein extraction
Protein assay
Protein labeling
2D Gel or 2D DIGE Gel
1 sample
2 samples
Gel images from scanning (no staining)
Left: 1 CyDye labeling
Right: 2 CyDye labeling
Gel transfer to Membrane
1 sample
2 samples
Membrane Image from fluorescence scanning (no staining): transfer efficiency is ~100%
Fluorescent 2D Western Blot
1-color Western Blot
2-color Western Blot
2D Western Blot
Spot matching
Stripping /re-blotting
Protein ID by Mass Spectrometry
2D Western blot procedure summary (1-color and 2-color):
- Sample prep: Protein from mouse liver samples were extracted and exchanged into 2D lysis buffer.
- 2D DIGE: Proteins was labeled, followed by IEF (pH 4-7 linear) and 2D according to Applied Biomics’ protocol.
- Gel image scan: Gel images were scanned after 2D electrophoresis (no staining).
- Transfer: Proteins from 2D gel were transferred to membrane.
- Membrane image scan: Membrane images were scanned after transfer.
- 1-color and 2-color Western blot:
- 1-color Western blot (left): Keratin 8 (blue) and its posttranslational modified forms.
- 2-color Western blot (right): Keratin 8 (red), Keratin 18 (green) and their posttranslational modified isoforms.
- Strip and Re-blot: The 2D membrane was stripped, re-blocked, and re-blotted to reveal beta-actin (green).
Application Gallery
Each standard 2D Western blot contains 1 sample blotted against 1 antibody. However, our 2D DIGE Western Blot allows 2 samples blotted against 1 antibody or 1 samples blotted against 2 antibodies in each Western blot, which generates more data at a lower cost.
1. 2D DIGE Western Blot: Protein Profiling & Antibody Detection In 1 Gel
Here we present a 2D DIGE gel with 2-samples, followed by Western Blot (WB). This SINGLE gel allows comparisons of protein profiling between the Control and Treated sample by 2D DIGE AND antibody detection by WB. Such analysis is only feasible using 2D DIGE Western Blot.
Step 1: Proteins were extracted from the Control and Treated Mouse liver samples, followed by 2D DIGE (pH4-7) using Applied Biomics’ protocols. The data was processed by In-gel Analysis.
DIGE Gel image: Control
DIGE Gel image: Treated
Control / Treated
Step 2: Proteins from 2D DIGE gel were transferred to the membrane and membrane images were scanned.
Step 3: Western Blot was performed using anti-keratin 8 & keratin 18 antibody as primary antibodies, and CF555-labeled goat anti-rabbit Ig (Biotium, Hayward, California) as secondary antibody. The Western Blot image revealed K8 and K18 spots.
Membrane Image: Control
Membrane Image: Treated
WB Image: K8 and K18 Spots
2. Fluorescent 2-Color Western Blot
This 2-color WB is feasible if the two primary antibody are generated in different animal species. The following example shows mouse liver proteome blotted by 2 different antibody. A. 2D gel image of mouse liver proteome; B. Fluorescent 2-color Western blot using rabbit anti-keratin 8 and mouse anti-keratin 18 as primary antibodies followed by CF 555-labeled goat anti-rabbit Ig (Biotium, CA) and CF647-labeled goat anti-mouse Ig (Biotium, CA) as a secondary antibodies.
A: Protein Image
B: Double-color Western Blot
