NHD provides since 2008 its Proteomics services to research institutes and industry. In contrast to commonly used peptide based analyses, we offer analyses of intact proteins which may include all information about
- • protein isoforms
- • splicing variants
- • post translational modifications such as
phosphorylation, glycosylation or oxidation
- • pI
- • molecular weight
Our longterm expertise, the usage of validated SOPs, our state of the art fluorescence technologies and equipment ensure you high quality analysis at a very competitive price. This includes analyses of human and animal tissue and fluids as well as microbial and plant cells and extracts.
2DE Analyses (selection)
- • Refraction-2D (Differential Protein Analysis)
- • Saturn-2D (Differential Analysis of Scarce Samples)
- • Saturn-2D REDOX (RedOx Status Analysis)
- • T-Rex 2D-PHOS (Phosphorylation Analysis)
- • T-Rex 2D-GLYCO (Glycosylation Analysis)
- • T-Rex 2D WB (2D Western Blot Analysis)
- • T-Rex 2D HCP (Host Cell Protein Analysis)
Depending on the sample and the aim of the analysis different technologies may be ideally suited for highly sensitive protein detection and precise quantification.
That is for two-dimensional differential gel electrophoresis (2D-DIGE):
- • Refraction-2D™ for the quantitative 2D gel analysis of different proteomes or protein samples,
- • Saturn-2D™ for quantitative 2D gel analysis of different proteomes of scarce samples,
- • Saturn-2D™ REDOX that illustrates changes within the complex redox interaction network between different samples.
- • T-Rex combined with a phospho-stain for differential protein phosporylation studies
- • T-Rex combined with a glyco-stain for differential protein glycosylation studies
- • T-Rex combined with Western Blotting for e.g. HCP analysis
For standardized and absolute quantification of proteins in 1D gels (SDS-PAGE, SAR-PAGE, IEF) as well as for standardized and for quantitative Western Blots we are using the
- • proteome studies
- • differential protein expression analysis (2D-DIGE)
- • post translational protein modifications
- • biomarker identification
- • 2D Western blotting
- • HCP coverage 2D Western blotting
- • standardized and quantitative SDS- and SAR-PAGE, IEF and
Western Blots of up to 52 samples per run
Host Cell Protein
2D Western Blot Analysis
For the production of biopharmaceuticals using host protein expression systems the identification and removal of host proteins is crucial.
The fluorescence-label based 2D Western Blot is the most powerful method for the analysis of host cell proteins (HCP). Here, all cell proteins are pre-labeled with the T-Rex fluorescent label, seperated by 2D electrophoresis and then transfered to a Western Blot membrane.
The following Western Blot analysis and fluorescence/ chemiluminescence detection allows to presicely discriminate the overexpressed proteins from the host cell proteins by anti-HCP antibodies.
The HCP coverage analysis is then automatically performed by Delta2D analysis software.
|pI (isoelectric point)||
and or pI
and or pI
|PTM for e.g.||
activation/ deactivation of kinases
signal transduction of tumors,
signal transduction upon pathogen reception
|metabolism of tumors
important role in Alzheimers desease, diabetis, ateriosclerosis
activation/ deactivation of proteins
|PubMed Publications 2016||13,716||2,300||1,149|
|Pubmed Publications until May 2017||6,834||1,150||543|
Two-dimensional gel electrophoresis
The principle of 2DE
The two-dimensional gel electrophoresis (2-D electrophoresis, 2-DE) is the most powerful method to separate complex protein mixtures.
It is based on the separation of intact proteins (not peptides) according to two molecular properties: the electric charge and the size of a protein.
The 2-D electrophoresis starts with the first-dimension separation - proteins are separated according to their electric charge, that is their isoelectric point. This step is called isoelectric focusing (IEF).
The IEF is (now a days) performed on a IEF strip, an acrylamide strip containing a pH gradient. When an electric potential is given to the IEF strip, the proteins will move along the strip to that point where their electric charge is 0 (neutral charge). After the IEF, the IEF strip now holds “a line of” the separated proteins according to their electrical charge.
After a step of equilibration the IEF strip is then applied to a SDS-PAGE gel in order to run the second dimension. This separation step now separates the now denatured proteins by their molecular weight.
2DE separation capacity
The protein separation capacity is given by
- a) the capacity of protein load of the IEF strip
- b) the separation area of the second dimension (the size of the SDS-PAGE gel).
For protein extracts from E. coli one should separate about 500 different proteins (e.g. detected by T-Rex fluorescent protein label) with 7 cm IEF strips and mini gel size SDS-PAGE (8 x 10 cm) and about 1200 (e.g. detected by T-Rex fluorescent protein label) proteins when using 24 cm strips and maxi size SDS-PAGE (27 x 22 cm).
Protein detection/ visualization
The number of proteins which can be detected is dependent on the method of protein visualization.
The general sensitivity limit is...
- • for Coomassie stains: a protein with an abundance of in the range of 100-10 ng can be detected.
- • for silver nitrate stains: a protein with an abundance of about 10-1 ng can be detected.
- • for fluorescence protein labels (minimal lysine labeling): proteins with an abundance of about 1 – 0.1 ng can be detected*.
- • for fluorescence protein labels (saturation cysteine labeling): proteins with an abundance of about 0.1 – 0.01 ng can be detected*.
* adequate fluorescence detection imaging device required
Differential Gel Electrophoresis/ Sample multiplexing
The usage of fluorescent protein labels is a strong benefit of 2D gel electrophoresis as the detection limit and dynamic range of the signal is much better and the analysis does not suffer from protein staining artifacts like background issues, difference in results due to room temperature, freshness of stains, etc… .
However, as proteins carrying a covalently bound fluorescence tag (label), they can be specifically detected. This allows to combine many samples to one 2DE analysis to allow for a precise quantification of protein expression and/or the accurate detection of post-translational protein modifications and to dramatically reduce the number of required 2D gels.
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Analysis "Get an Idea Level"
Rapid proof of concept analysis to evaluate possibilities
Analysis/ Study "Publication Level"
Experiments/ studies for identification of significantly regulated proteins
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