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2D DIGE Western Blot Analysis: Protein Profiling & Antibody Detection In 1 Gel


 

 

 

Here we present a 2D DIGE gel with TWO 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

2D DIGE & Western Blot

DIGE Gel image: Treated

2D DIGE & Western Blot

Control / Treated

2D DIGE & Western Blot

 

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 and keratin 18 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

2D DIGE & Western Blot

Membrane Image: Treated

2D DIGE & Western Blot

Western Blot Image: K8 and K18 Spots

2D DIGE & Western Blot