Navigation Links
Detection and quantitation of low-abundant proteins with ECL Plex fluorescent Western blotting

O. Rönn1, M. Landström2, A. Edman-Örlefors1, and S. Edlund1
1 GE Healthcare, Uppsala, Sweden
2 Ludwig Institute for Cancer Research, Uppsala, Sweden


ECL Plex™ Western Blotting Detection System can detect proteins at picogram levels, with linearity over 3.6 orders of magnitude. Two antigens can be detected simultaneously, and differential quantitation of low-abundant proteins, such as phosphoproteins in cell lysates, can be performed.


Introduction
ECL Plex Western blotting detection system is based on direct fluorescent light detection from CyDye™ labeled antibody conjugates. The use of different fluorophores, Cy3 and Cy5, enables simultaneous detection of multiple antigens on the same blotted membrane (Fig 1). This eliminates possible loss of material from a stripping procedure, providing more accurate data. Together with a high-end imaging system (e.g. Typhoon™ scanner or Ettan™ DIGE Imager), this results in high linearity and broad dynamic range for superior detection and quantitation of low-abundant proteins.


Methods
Pure proteins or cell extracts were run on 12% Trisglycine gels. After electrophoresis, gels were blotted onto a Hybond™ ECL or Hybond-LFP membrane, and then probed with primary antibodies and secondary CyDyeconjugated antibodies according to the manufacturer’s instructions. Please see the application note Multiplex protein detection using the ECL Plex fluorescent Western blotting system (28-4015-40) (1), available at www.amershambiosciences.com, for detailed methods.


Results and discussion
Sensitivity and dynamic r ange
A model system, with two-fold serial dilutions of human apotransferrin and actin (not shown), was used to optimize system performance (Fig 2). Protein could be detected down to 1.2 pg, giving linearity over 3.6 orders of magnitude.

This result was also dependent on a membrane with low background fluorescence at wavelengths suitable for the fluorophores. Both a nitrocellulose membrane (Hybond ECL) and a newly developed PVDF membrane (Hybond–LFP) were found to meet these expectations.

Detection and quantitation of a phosphoprotein
Multiplex detection of proteins often suffers from cross-reactive signals between antibodies or dyes. This is addressed with the ECL Plex system through thorough optimization of components and protocols (Fig 3).

TGF-βis a potent growth factor stimulating a number of cellular responses including growth inhibition, cell differentiation, and apoptosis. The TGFβ–mediated phosphorylation of p38, measured in human T293 epithelial kidney cells, showed proportional increase when compared and quantitatively related to actin (Fig 4).


Conclusions
Western blotting is a well-established technique. The most common application is based on chemiluminescence, for example the GE Healthcare ECL™ product line (2, 3). As a new member in that family, ECL Plex adds a broader dynamic range and the ability to detect more than one protein on the same blot with high sensitivity and specificity. These features complement the chemiluminescence-based products, offering better quantitative data and shorter processing time. CyDye technology also offers longer lasting signals for storage and rescanning. Low-abundant proteins are readily detected in cell lysates, and activation of signal transduction pathways can be measured.


References
1. Application note: Multiplex protein detection using the ECL Plex fluorescent Western blotting system, GE Healthcare, 28-4015-40, Edition AA (2005).

2. Harper, D. R. et al. Protein blotting: ten years on. J. Virol. Methods 30, 25–39 (1990).

3. Paladichuk, A. How the Western was won: a profile of tools and kits available for Western blotting. The Scientist 13, 18–21 (1999).



back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A New PCR-based Mycoplasma Detection Method
3. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
4. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
5. Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes
6. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
7. The DIG System Nonradioactive and Highly Sensitive Detection of Nucleic Acids
8. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
9. Quantification of Nucleosomes in Serum by the Cell Death Detection ELISAplus
10. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
11. In Situ Cell Death Detection Kit
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/24/2016)... ... 2016 , ... Researchers at the Universita Politecnica delle Marche in Ancona combed ... pleural mesothelioma. Their findings are the subject of a new article on the Surviving ... signposts in the blood, lung fluid or tissue of mesothelioma patients that can help ...
(Date:6/23/2016)... Wausau, WI (PRWEB) , ... June 23, 2016 ... ... probiotic supplements, is pleased to announce the launch of their brand, UP4™ Probiotics, ... supplements for over 35 years, is proud to add Target to its list ...
(Date:6/23/2016)... 23, 2016 /PRNewswire/ - FACIT has announced the ... biotechnology company, Propellon Therapeutics Inc. ("Propellon" or ... of a portfolio of first-in-class WDR5 inhibitors for ... as WDR5 represent an exciting class of therapies, ... medicine for cancer patients. Substantial advances have been ...
(Date:6/23/2016)... , June 23, 2016 Houston Methodist ... the Cy-Fair Sports Association to serve as their ... agreement, Houston Methodist Willowbrook will provide sponsorship support, ... connectivity with association coaches, volunteers, athletes and families. ... the Cy-Fair Sports Association and to bring Houston ...
Breaking Biology Technology:
(Date:6/22/2016)... 22, 2016  The American College of Medical Genetics and ... Magazine as one of the fastest-growing trade shows during ... the Bellagio in Las Vegas . ... of growth in each of the following categories: net square ... number of attendees. The 2015 ACMG Annual Meeting was ranked ...
(Date:6/22/2016)... 22, 2016 On Monday, the Department of ... to share solutions for the Biometric Exit Program. The ... Border Protection (CBP), explains that CBP intends to add ... the United States , in order to ... imposters. Logo - http://photos.prnewswire.com/prnh/20160622/382209LOGO ...
(Date:6/20/2016)... , June 20, 2016 Securus ... justice technology solutions for public safety, investigation, corrections ... the prisons involved, it has secured the final ... (DOC) facilities for Managed Access Systems (MAS) installed. ... additional facilities to be installed by October, 2016. ...
Breaking Biology News(10 mins):