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 range
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:
(Date:3/25/2015)... 2015 /PRNewswire/ - SQI Diagnostics Inc. ("SQI" or the "Company") ... of recent discussions with certain shareholders representing approximately 45% ... after dealing with certain matters including the appointment of ... on March 26, 2015 (the "Meeting") it intends to ... will take place when the Meeting reconvenes. The Meeting ...
(Date:3/25/2015)... NEW YORK , March 25, 2015 /PRNewswire/ ... has commenced Factual Stock Report coverage on ... Bioceuticals (OTCQX: ATTBF; CSE: ATT): is a specialty ... subsidiaries of cultivating, licensing and marketing proprietary ingredients, ... medicinal markets in North America ...
(Date:3/25/2015)... CAMBRIDGE, Mass. and REYKJAVIK, ... which provides comprehensive capabilities for using the genome to create ... their publication of the largest studies of whole-genome data yet ... in Nature Genetics – are built ... variation, authored by a team of deCODE scientists led by ...
(Date:3/25/2015)... San Francisco, CA (PRWEB) March 25, 2015 ... Provide Support for Novel Mechanism of Action in ... March 25, 2015 – Cytokinetics, Incorporated (Nasdaq: CYTK) ... its fast skeletal muscle troponin activator tirasemtiv in ... from a Phase IIa “Evidence of Effect” or ...
Breaking Biology Technology:SQI Diagnostics Inc. Announces New Nominees to SQI Diagnostics Board and Adjournment of Meeting 2SQI Diagnostics Inc. Announces New Nominees to SQI Diagnostics Board and Adjournment of Meeting 3SQI Diagnostics Inc. Announces New Nominees to SQI Diagnostics Board and Adjournment of Meeting 4SQI Diagnostics Inc. Announces New Nominees to SQI Diagnostics Board and Adjournment of Meeting 5S&P Capital IQ Initiates Coverage on Abattis Bioceuticals Corp. in S&P Capital IQ Factual Stock Reports 2S&P Capital IQ Initiates Coverage on Abattis Bioceuticals Corp. in S&P Capital IQ Factual Stock Reports 3S&P Capital IQ Initiates Coverage on Abattis Bioceuticals Corp. in S&P Capital IQ Factual Stock Reports 4S&P Capital IQ Initiates Coverage on Abattis Bioceuticals Corp. in S&P Capital IQ Factual Stock Reports 5Landmark deCODE Studies Demonstrate the Power of Truly Big Genomics for Improving Medicine Worldwide 2Cytokinetics Announces Publication of Results from Phase II Trial of Tirasemtiv in Patients with Myasthenia Gravis 2Cytokinetics Announces Publication of Results from Phase II Trial of Tirasemtiv in Patients with Myasthenia Gravis 3Cytokinetics Announces Publication of Results from Phase II Trial of Tirasemtiv in Patients with Myasthenia Gravis 4Cytokinetics Announces Publication of Results from Phase II Trial of Tirasemtiv in Patients with Myasthenia Gravis 5Cytokinetics Announces Publication of Results from Phase II Trial of Tirasemtiv in Patients with Myasthenia Gravis 6
... SCOTTSDALE, Ariz., June 23 MedPredict has ... strategic insight for pharma and,biotech companies hoping ... pain,management. The report, entitled "Thought Leader Insight ... conducted with a panel of,specialists in anesthesiology, ...
... enhance industry leader,s dermatological portfolio, CORAL ... Inc.,the world,s largest independent pharmaceutical company specializing ... definitive merger agreement,pursuant to which Stiefel Laboratories ... BTRX ), through a two-step,transaction, a ...
... Concuity,s Product Suite by Adding Price Transparency, ... /PRNewswire-FirstCall/,-- Concuity, a healthcare division of Trintech (Nasdaq: ... management and revenue recovery technology ("Peer,Reviewed by HFMA") ... Systems (FHS Corp), a provider of a web-based ...
Cached Biology Technology:In Pain Market, Huge Unmet Needs, Tough Regulatory Environment Challenge Pharmaceutical Companies, According to MedPredict 2Barrier Therapeutics to Merge Into Stiefel Laboratories 2Barrier Therapeutics to Merge Into Stiefel Laboratories 3Barrier Therapeutics to Merge Into Stiefel Laboratories 4Barrier Therapeutics to Merge Into Stiefel Laboratories 5Concuity Announces Partnership with Financial Healthcare Systems, LLC 2Concuity Announces Partnership with Financial Healthcare Systems, LLC 3Concuity Announces Partnership with Financial Healthcare Systems, LLC 4
(Date:3/12/2015)... , March 12, 2015 WHEN:Tuesday, March 17, ... here: http://bit.ly/1G7Os0L . SPEAKERS: , Frost & ... - http://photos.prnewswire.com/prnh/20150311/181111 Biometric companies ... compete in several different markets and industries. This ... witnessing an uptrend. Join Frost ...
(Date:3/10/2015)... , March 10, 2015  Continuing its 167-year history ... Hammacher Schlemmer introduces The Eye Scanning Password Authenticator ... granting access to secure websites or sensitive data. ... travelers at international borders, the device has a small ... points of the iris, converting them into an encrypted ...
(Date:3/10/2015)...  NXT-ID, Inc. (NASDAQ: NXTD ... authentication company focused on the growing mobile commerce ... been named the "Number One Best Tech Gift ... Modern ( http://www.rethinkmodern.com/ ) is a web magazine focused ... that have a unique function or design.  ...
Breaking Biology News(10 mins):Biometrics Key to Future Growth in Healthcare, Retail and Financial Markets 2Biometrics Key to Future Growth in Healthcare, Retail and Financial Markets 3Hammacher Schlemmer Introduces The Eye Scanning Password Authenticator 2NXT-ID's Wocket Smart Wallet Named Number One Best Tech Gift for 2015 by Rethink Modern 2NXT-ID's Wocket Smart Wallet Named Number One Best Tech Gift for 2015 by Rethink Modern 3NXT-ID's Wocket Smart Wallet Named Number One Best Tech Gift for 2015 by Rethink Modern 4
... potential new treatment for alcoholism called nalmefene is effective ... individuals, says a new study published this week in ... regarded as the primary treatment goal for alcohol dependence, ... for relapse prevention. However, relapse rates remain high and ...
... ( Journal of Visualized Experiments ) will publish a ... phone camera can capture images from a fluorescent microscope and flow ... to easily run tests such as checking for contaminated water and ... article electrical and bio engineers from the University of California Los ...
... biodiversity observation network could be established with modest funding ... in the May 2013 issue of BioScience . ... in scientists, understanding of the global distribution of marine ... pollution, habitat destruction, and overfishing. The network would help ...
Cached Biology News:A new treatment option for alcohol dependence: Reduced consumption rather than abstinence 2Cell phone camera photographs microscopic cell samples 2
... Cellject Uno Electroporator is a simple, ... optimum efficiencies in bacterial and yeast ... one of two voltages and either ... mm cuvettes. On pressing the pulse ...
Request Info...
Maxi XL Hybridization Oven for Extra Large bottles. 6 bottle (70 mm) rotisserie + shaker tray available for simultaneous use. Includes linear and orbital shaker trays, 4 extra large bottles, pack o...
Shake 'n' Stack oven - New Model (includes 10 bottle capacity rotisserie, adjustable feet, drip tray, manual)...
Biology Products: