2D DIGE Proteomics Service

2D gel electrophoresis involves isoelectric focusing (IEF) as 1^st dimension and SDS-PAGE as 2^nd dimension, separating proteins by both pI (in IEF) and molecular weight (in SDS-PAGE). 2D-DIGE proteomics analysis is an improved version of standard 2D gel electrophoresis. In addition to all features of 2D gel, 2D DIGE labels different samples with up to 3 fluorescent tags, offering significant advantages that standard 2D gel electrophoresis & other proteomics techniques cannot achieve. Please view here for comparisons of experimental procedures between 2D-DIGE and 2D gel electrophoresis.

Furthermore, our 2D DIGE proteomics services employ large format 2D gel electrophoresis, customized sample preparations and versatile study designs, offering a powerful platform for the following applications:

Identifying therapeutic or diagnostic markers in different diseases
Identifying key regulators in major biological pathways
Studying post-translational modifications such as phosphorylation, glycosylation, etc.
Determining HCP antibody coverage and detecting HCP contaminants

2D DIGE experimental procedure

2D DIGE & mass spectrometry full procedure

Advantages of 2D DIGE over 2D Gel Electrophoresis:

Higher sensitivity: 2D DIGE uses Fluorescent labeling with a sensitivity of 0.2 ng/spot, versus the sensitivity of standard 2D gel electrophoresis with Coomassie blue staining at 100 ng/spot or silver staining at 1 ng/spot
Higher accuracy: The extremely high spot resolution enables accurate spot quantitation. Differences in protein expression as small as 10% can be detected
Higher reproducibility: Nearly identical data was obtained on the same sample labeled with different CyDye on the same gel or cross different gels, thus eliminating the need of running technical replicates
Broader spectrum: Our large 2D DIGE gel format resolve ~5000 protein spots, allowing a wide dynamic range detection/quantitation of low abundant proteins, large proteins and small peptides
Fewer number of gels: Each 2D DIGE gel can accommodate up to 3 different samples, versus 1 sample on each 2D Gel. For the same number of samples, only 1/3 number of 2D DIGE gels is needed.
Lower cost: Our price is based on each gel instead of each sample. Lower number of 2D DIGE gels and no need of technical replicates translates into higher quality data at a lower cost than standard 2D Gel service. Price covers from sample preparation to publication-ready data.
Fast turn-around: Results In 5-7 Days.

2D DIGE experimental procedure:

Sample preparation: Proteins are extracted following customized sample preparation protocols. Protein concentrations are determined by protein assay and adjusted to the desired concentration.
Sample labeling with fluorescent dyes: Equal amount of protein extract from each sample is labeled by fluorescent dyes (size and charge matched). The spectrally resolvable dyes enable simultaneous co-separation and analysis of up to 3 samples on a single multiplexed gel.
2D gel electrophoresis: Up to 3 samples can be simultaneously separated on a single 2D gel, with isoelectric focusing (IEF) in the 1st dimension and SDS-PAGE in the 2nd dimension.
Image acquisition: After 2D gel electrophoresis, the gel is scanned using a Typhoon image scanner. Each scan reveals one of the CyDye signals (Cy2, Cy3 and Cy5).
Image analysis: ImageQuant software is used to generate the image data including the image of individual sample and overlay image of 2 or 3 samples.
Quantitative analysis: Comparative analysis of all spots using DeCyder “in-gel” or “cross-gel” analysis software. The protein expression ratios between different samples or different groups of samples are generated.
Automated spot picking: Following the spot picking design using DeCyder software, protein spots of interest can be automatically picked from the 2D gel with the Ettan Spot Picker, followed by Protein Identification by Mass Spectrometry.
Cluster Analysis: Proteins are analyzed in clusters according to their functions. This is a complimentary service for projects with 50 or more identified proteins.
Validation: We offer 2D Western Blot services to validate the 2D DIGE data.

View a sample data report

2D DIGE Images for various Sample Types

Human Cells		Human Fluid/Tissue	Lab Animals	Bacteria & Others
Jurkat cells	Breast cancer cell line	Saliva	Mouse & Rat brain	E. coli
PBMCs	Beta-islet	Urine	Mouse oocyte	Salmonella
HEK293	Hepatocytes	Amniotic fluid	Mouse retina cells	Bacteria-secreted
Blood T cells	Keratinocytes	Adipose	Rat tear	Yeast
Blood monocyte	Platelets	Liver tissue	Drosophila	Plant
Dendritic cells	Blood cell exosome	Serum / Plasma	C. elegans	Rice
		Cerebrospinal fluid	Zebrafish	Flu vaccines

Jurkat cells

2D image of jurkat cell proteome

Human Jurkat cells (pH 3-10 NL)

Human PBMCs

2D image of human PBMC proteome

Human PBMC (pH 3-10)

HEK293 cells

2D image of HEK293 cell proteome

HEK293 cell (pH 3-10 L)

Human blood T cells

2D image of human blood T cell proteome

Human blood T cell (pH 3-10)

Human blood monocytes

2D image of human blood monocyte proteome

Human blood monocyte (pH 3-10)

Human dendritic cells

2D image of human dendritic cell proteome

Human dendritic cell (pH 3-10)

Human breast cancer cell line

2D image of human breast cancer cell line MCF7

Human breast cancer cell line (pH 3-10)

Human beta-islet cells

2D image of human beta-islet cell proteome

Human beta-islet cell (pH 3-10)

Human hepatocytes

2D image of human hepatocyte proteome

Human hepatocyte (pH 3-10)

Human keratinocytes

2D image of human keratinocyte proteome

Human keratinocyte (pH 3-10)

Human platelets

2D image of human platelet proteome

Human platelet (pH 3-10 L)

Human blood cell exosome

2D image of human blood cell exosome proteome

Human blood cell exosome (pH 3-10)

Human saliva

2D image of human saliva proteome

Saliva (pH 4-7)

Saliva (pH 3-10)

2D image of human saliva proteome pH 3-10

Human urine

2D image of human adult urine proteome

Urine (pH 4-7)

Urine (pH 3-10)

2D image of human adult urine proteome pH 3-10

Human amniotic fluid

2D image of human amniotic fluid proteome

Human amniotic fluid (pH 3-10)

Human adipose

2D image of human adipose proteome

Human adipose proteome (pH 3-10)

Human liver tissue

2D image of human liver tissue proteome

Human liver tissue (pH 3-10)

Human serum

2D image of human serum proteome. See our serum proteomics page for more info.

Serum (pH 4-7)

Serum (pH 3-10)

2D image of human serum proteome pH 3-10

Human cerebrospinal fluid

2D image of human cerebrospinal fluid proteome

Human cerebrospinal fluid (pH 3-10)

Mouse & Rat Brain

2D images of mouse & rat brain proteome

Mouse brain (pH 3-10)

Rat brain (pH 3-10)

Mouse oocyte

2D image of mouse oocyte proteome

Mouse oocyte (pH 3-10)

Mouse retina cells

2D image of mouse retina cell proteome

Mouse retina cell (pH 3-10)

Rat tear

2D image of rat tear proteome

Rat tear (pH 3-10)

Drosophila

2D image of drosophila proteome

Drosophila (pH 3-10 NL)

C. elegans

2D images of C. elegans proteome

C. elegans (pH 4-7)

C. elegans (pH 3-10)

Zebrafish

2D image of zebrafish proteome

Zebrafish (pH 3-10 L)

E. coli

2D image of E. coli proteome

E. coli (pH 3-10 NL)

E. coli (pH 3-10 NL)

2D black-white image of E. coli proteome

Salmonella

2D image of salmonella proteome

Salmonella (pH 3-10 L)

Bacteria-secreted proteins

2D image of bacteria-secreted proteins proteome

Bacteria-secreted proteins (pH 3-10)

Yeast

2D image of yeast cell proteome

Yeast (pH 3-10 NL)

Yeast (pH 3-10 NL)

2D black-white image of yeast cell proteome

Plant

2D image of plant (N. benthamiana) proteome

N. benthamiana (pH 3-10)

Rice

2D image of rice proteome

Rice (pH 3-10)

Flu Vaccines

2D image of flu vaccines from different manufacture lots

Manufacturer lot 1

Manufacturer lot 2

M1 / M2

2D DIGE FAQs

Frequently asked questions about 2D-DIGE advantages, process, time & cost:

Compared to 2D-gel, 2D-DIGE offers higher sensitivity, higher accuracy, fewer number of gels, faster turn around, and a lower price.

Learn the difference

The sensitivity of 2D DIGE is about 0.2 ng/spot.

2D DIGE is very accurate due to the extremely high spot resolution. Differences in protein expression as small as 10% can be detected.

Highly reproducible: identical data can be obtained on the same sample labeled with different CyDye on the same gel or cross different gels (see an example).

2000-5000 protein spots using pH3-10 IPG strip. If using additional IPG strips such as pH 4-7, pH 6-9 and pH 7-11, over 5000 spots can be detected.

Most customers request to start the experiment with an Analytical Gel for the following reasons:

Analytical gels use about 25 µg of each sample while prep-gels use about 250 µg of each sample. The total protein load for analytical gel is about 75 µg, while total protein load for prep-gels is about 750 µg. Since Analytical Gel contains lower sample amount resulting in less ion and salt in IEF, it will provide more accurate quantitation of protein changes) and much higher image quality for publication/presentation purposes.
After running the analytical gel, the in-gel data analysis will be done. Based on the analytical gel results, you can put more control or treatment samples if your desired spots are from control (usually down-regulation), or from treatment (usually up-regulation or newly induced proteins). In contrast, Prep-gel is much less likely to provide such information due to the excess amount of protein.
From the analytical gel, you will know if you need to focus on a specific region (i.e. with most changes) in the next 2-D experiment by changing the pH range for IEF or the percentage of the SDS-gel (for resolving lower or higher MW protein spots). In contrast, Prep-gel is much less likely to provide such information due to the excess amount of protein.
The main purpose of running Prep-gel is to obtain enough protein amount for the protein ID by Mass Spectrometry.

5-7 days for Analytical gel.

7-9 days for Prep-gel, spot picking and protein identification by mass spectrometry.

The data report contains two main parts:

ImageQuant analysis: gel images of individual samples and overlay of two sample images
DeCyder analysis: quantitative analysis and comparison of all spots between different samples.

Yes, we can send you the raw data generated by instrument software free of charge within 1 year of the completion of project. If you request raw data after 1 year, it may incur a charge based on the actual time to retrieve them from achieve.

Let’s assume a new customer from academia is interested in finding the differential protein expression among 2-3 samples, followed by obtaining Protein ID for the top 10 spots. Please note that the prices are per gels, not per sample. Each gel can load up to 3 samples. Following would be the estimated cost:

Service Description	2 samples	3 samples
2D DIGE Analytical Gel	$1050*	$1195*
2D DIGE Preparative Gel	$1200	$1349
Spot Picking (up to 96 spots per gel)	$265	$265
10 Protein ID by Mass Spectrometry	$1390	$1390
Total cost	$3905	$4199

* Only applies for new customers who submit standard protein samples with protein concentration >5mg/ml. The discount is subject to approval from Finance. The discounted price applies for the first 2D DIGE Analytical gel. The discounted price does NOT apply for Preparative gels.