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 could achieve. The fluorescent labeling also allows accurate quantitation of WB signals. This platform has been successfully applied in the following areas:

  1. Identify protein spots interacted with antibody
  2. Study HCP antibody coverage
  3. 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 Blot2D DIGE Western Blot
Number of gels for each AbTwoOne
Detection MethodSilver Staining *CyDye Labeling *
Protein and WB in same gelImageImage
In-gel protein and WB comparisonImageImage
AccuracyLowHigh
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 ReproducibilityLowerHigher
* Sensitivity of fluorescent CyDye labeling is higher than that of silver staining (view details)

2D Western Blot experimental procedure

2D Western Blot procedure: step 1 - tissues or cells or any biological samples as starting material

Tissues or cells

2D Western Blot procedure: step 2 - protein extraction from mouse liver and labeling with fluorescent dyes

Protein extraction
Protein assay
Protein labeling

2D Western Blot procedure: step 3 - IEF and SDS-PAGE

2D Gel or 2D DIGE Gel

1 sample

2D Western Blot procedure: step 4A - Gel image scan with 1 CyDye labeling

2 samples

2D Western Blot procedure: step 4B - Gel image scan with 2 CyDye labeling

Gel images from fluorescence scanning (no staining).
Left: DIGE with 1 CyDye labeling
Right: DIGE with 2 CyDye labeling

2D Western Blot procedure: step 5 - transfer proteins from gel to membrane

Gel transfer to Membrane

1 sample

2D Western Blot procedure: step 6A - Membrane image scan with 1 CyDye labeling

2 samples

2D Western Blot procedure: step 6B - Membrane image scan with 2 CyDye labeling

Membrane Image from fluorescence scanning (no staining): transfer efficiency is ~100%)

Fluorescent 2D Western Blot

1-color Western Blot

2D Western Blot procedure: step 7A - image of 1-color Western Blot with anti-Keratin 8

2-color Western Blot

2D Western Blot procedure: step 7B - image of 2-color Western Blot with anti-Keratin 8 and 18

2D Western Blot

Spot matching

2D Western Blot procedure: step 8 - Matching Western Blot spots to spots on gel image

Stripping /re-blotting

2D Western Blot procedure: step 9 - Membrane stripping and re-blotting with anti-actin antibody

Protein ID by Mass Spectrometry

2D Western Blot procedure: step 10 - identify reactive spots by mass spectrometry

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

  1. Sample prep: Protein from mouse liver samples were extracted and exchanged into 2D lysis buffer.
  2. 2D DIGE: Proteins was labeled, followed by IEF (pH 4-7 linear) and 2D according to Applied Biomics’ protocol.
  3. Gel image scan: Gel images were scanned after 2D electrophoresis (no staining).
  4. Transfer: Proteins from 2D gel were transferred to membrane.
  5. Membrane image scan: Membrane images were scanned after transfer.
  6. 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.
  7. 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
  2. Fluorescent 2-Color Western Blot

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

2D DIGE Western Blot: Control sample proteome gel image

DIGE Gel image: Treated

2D DIGE Western Blot: Treated sample proteome gel image

Control / Treated

2D DIGE Western Blot: Overlay image of Control and Treated proteome

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

2D DIGE Western Blot: Control sample proteome membrane image

Membrane Image: Treated

2D DIGE Western Blot: Treated sample proteome membrane gel image

WB Image: K8 and K18 Spots

2D DIGE Western Blot: Control and Treated blotted with Anti-K8/K18 antibody

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

Fluorescent 2-Color Western Blot: Mouse liver proteome image

B: Double-color Western Blot

Fluorescent 2-Color Western Blot: Mouse liver blotted with anti-K8 in green and anti-K18 in red

FAQ

Frequently asked questions about 2D Western Blot advantages, process & time:

Our DIGE WB only needs 1 gel vs. 2 gels in the standard WB, which translates into lower cost. It allows the direct visualization of antibody coverage that the standard WB cannot. In addition, the fluorescent labeling (instead of silver staining) offers higher sensitivity, accuracy and consistency.

2D DIGE WB is the only platform that allows high sensitivity visualization and accurate quantitation of protein PTMs in 1 test, even from high complexity samples such as cell lysates and tissue. Due to the very low levels of PTM (1-2% of total proteins) in nature, it would require protein enrichment (such as fractionation or Immuno-precipitation) for LC-MS/MS to detect the PTM, which translates into higher cost and low accuracy.

Please refer to the ‘Sample info’ tab under each service.

7-9 days.

No.

Due to unique feature of DIGE platform, you can have 3 study designs:

  • 1 sample + 1 antibody
  • 1 sample + 2 antibody
  • 2 samples + 1 antibody