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2D Western Blot: From Samples To Data
More Examples on 2D Western Blot

 

 

 

Applied Biomics offers the most comprehensive antibody detection analysis by using high resolution large format 2D fluorescent Western Blot (WB). Our report provides Protein, WB and Protein/WB overlay images, allowing the direct visualization of antibody detection that no other platform could achieve. This platform has been successfully applied in the following areas:

1) Identify protein spots interacted with antibody
2) Study different post-translational modifications (PTMs) of your target proteins
3) Study HCP antibody coverage

The unique feature of our 2D DIGE Western Blot allows 3 study designs:

Study design 1: Sample-1, Western Blot
Study design 2: Sample-1, Sample-2, Western Blot (see an exmaple) - 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 only if the two primary antibody are generated in different animal species)

Our platform offers significant ADVANTAGES over ELISA and standard 2D Western blot:

1) High consistency: Fluorescent 2D Western detects both protein and WB images from the same gel and same membrane, allowing the perfect alignments between protein and WB spots, thus eliminating any gel-to-gel or gel-to-membrane variations as seen in the traditional 2D WBs.
2) Cost-effective: Since only one gel is needed, it saves the cost of running duplicate gels.
3) High accuracy: Fluorescent CyDye labeling allows accurate quantitation of protein and WB spots.
4) High sensitivity: Fluorescent CyDye labeling has a higher sensitivity and wider dynamic range over the silver staining.

 

Tissue or cells Tissues or cells

Protein extraction/assay/labeling


Protein extraction
Protein assay
Protein labeling


2D Gel or 2D DIGE Gel


2D Gel or 2D DIGE Gel
 
2D Gel Image (1 sample)

2D Gel Image (1 sample)
2D DIGE Gel Image (2 samples)

2D DIGE Gel Image (2 samples)
Image captured from the gel (no staining)
 


Gel transfer to Membrane

2D Membrane Image (1 sample)

2D Membrane Image (1 sample)

2D DIGE Membrane Image (2 samples)

2D DIGE Membrane Image (2 samples)
Image captured from 2D membrane (no staining)


 
Fluorescent 2D Western Blot  

2D Western Blot: one color


2D Western Blot: one color

2D Western Blot: two color

2D Western Blot: two color
2D Western Blot


 
Spot matching

Spot matching
Stripping and re-blotting with other antibodies

Stripping and re-blotting with other antibodies
 


Protein ID by Mass Spectrometry

Protein ID by Mass Spectrometry

   

 

2D Western blot (1-color and 2-color):

1) Mouse liver samples were prepared using 2D lysis buffer.

2) Proteins was labeled and analyzed by 2D DIGE using pH 4-7 linear IPG strip according to Applied Biomics protocol. Left: DIGE with 1 CyDye labeling; Right: DIGE with 2 CyDye labeling

3) The images were scanned after 2D electrophoresis, using Typhoon 9400; then the proteins from 2D gel were transferred to membrane.

4) 1-color and 2-color Western blot:

Left: 1-color Western blot to reveal Keratin 8 (blue) and its posttranslational modified forms. The secondary antibody is CF488-labeled goat anti-mouse IgG (Biotium, Hayward, California).

Right: 2-color Western blot to reveal keratin 8 (red), Keratin 18 (green) and their posttranslational modified isoforms. The secondary antibodies are CF555-labeled goat anti-rabbit Ig and CF647-labeled goat anti-mouse Ig (Biotium, Hayward, California).

5) The 2D membrane was stripped after scanning, re-blocked, followed by Western blot to reveal beta-actin.