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:8/1/2014)... 2014 Applied DNA Sciences, Inc. ... anti-counterfeiting technology and product authentication solutions, will host a ... its fiscal third quarter results. The Quarterly Report on ... the end of the day on Monday August 11, ... host a conference call beginning at 9:00 A.M. Eastern ...
(Date:8/1/2014)... , Aug. 1. 2014 As one of ... 2014 AACC Annual Meeting & Clinical Lab Expo is ... patient care are introduced to the healthcare world. This ... Expo,s 650 exhibitors will include the latest diagnostic technology ... Advances in smartphones and biosensors have enabled the development ...
(Date:8/1/2014)... , August 1, 2014 According ... Research "Biophotonics Market - Global Industry Analysis, Size, Share, ... was valued at USD 25.16 billion in 2013, which ... growing at a CAGR of 10.4% from 2014 to ... at  http://www.transparencymarketresearch.com/biophotonics-market.html Increasing demand for accurate ...
(Date:8/1/2014)... Aug. 1, 2014 Cambrex Corporation (NYSE: ... 30, 2014. Highlights , Second ... in the same period last year. , Second quarter ... in the same period last year (see table at the ... guidance increased, excluding the impact of foreign currency, to between ...
Breaking Biology Technology:Applied DNA Sciences to Host Conference Call Tuesday August 12, 2014 2Applied DNA Sciences to Host Conference Call Tuesday August 12, 2014 3Lab-on-a-Chip Technology to Be Featured at 2014 AACC Annual Meeting & Clinical Lab Expo 2Lab-on-a-Chip Technology to Be Featured at 2014 AACC Annual Meeting & Clinical Lab Expo 3Global Biophotonics Market is Expected to Reach USD 50.20 Billion in 2020: Transparency Market Research 2Global Biophotonics Market is Expected to Reach USD 50.20 Billion in 2020: Transparency Market Research 3Global Biophotonics Market is Expected to Reach USD 50.20 Billion in 2020: Transparency Market Research 4Cambrex Reports Second Quarter 2014 Financial Results 2Cambrex Reports Second Quarter 2014 Financial Results 3Cambrex Reports Second Quarter 2014 Financial Results 4Cambrex Reports Second Quarter 2014 Financial Results 5Cambrex Reports Second Quarter 2014 Financial Results 6Cambrex Reports Second Quarter 2014 Financial Results 7Cambrex Reports Second Quarter 2014 Financial Results 8Cambrex Reports Second Quarter 2014 Financial Results 9Cambrex Reports Second Quarter 2014 Financial Results 10Cambrex Reports Second Quarter 2014 Financial Results 11Cambrex Reports Second Quarter 2014 Financial Results 12Cambrex Reports Second Quarter 2014 Financial Results 13Cambrex Reports Second Quarter 2014 Financial Results 14Cambrex Reports Second Quarter 2014 Financial Results 15Cambrex Reports Second Quarter 2014 Financial Results 16
... Medical Technology. Inc.,has been awarded a five-year contract ... VHA Inc. and the University HealthSystem,Consortium (UHC), two ... The agreement makes Liftaem(TM) available to more ... "By using proper equipment such,as Liftaem(TM), hospitals not ...
... 12 VIA Pharmaceuticals,(Nasdaq: VIAP ), ... of,compounds for the treatment of cardiovascular disease, ... board. Joining the clinical advisory,board,s chair, Marc ... Medical School, and Senior Physician, Brigham & ...
... Breakthrough Pain Associated with Chronic Pain Conditions in ... ... Around-the-Clock, FRAZER, Pa., Nov. 12 Cephalon, Inc. (Nasdaq:,CEPH) ... the U.S. Food and Drug Administration (FDA) seeking approval to,market FENTORA(R) ...
Cached Biology Technology:Smart Medical Technology, Inc. Awarded Novation Contract for Ground-breaking Liftaem(TM) System that Aids Lifting and Transferring Patients 2VIA Pharmaceuticals Expands Clinical Advisory Board With Leading Experts in Cardiovascular Disease 2VIA Pharmaceuticals Expands Clinical Advisory Board With Leading Experts in Cardiovascular Disease 3VIA Pharmaceuticals Expands Clinical Advisory Board With Leading Experts in Cardiovascular Disease 4Cephalon Submits Supplemental New Drug Application for FENTORA 2Cephalon Submits Supplemental New Drug Application for FENTORA 3Cephalon Submits Supplemental New Drug Application for FENTORA 4Cephalon Submits Supplemental New Drug Application for FENTORA 5Cephalon Submits Supplemental New Drug Application for FENTORA 6Cephalon Submits Supplemental New Drug Application for FENTORA 7
(Date:8/1/2014)... 10,000 reptile species have been recorded into the ... reptile species and classification, making the reptile species ... world, alongside bird and fish species. , For ... mark the year that reptiles would become the ... include snakes, lizards, turtles, crocodiles, tuataras and amphisbaenians. ...
(Date:8/1/2014)... of the UC Davis Center for Neuroscience and ... Science and Engineering Faculty Fellowship from the U.S. ... will support new work on learning and memory ... , The new project aims to connect neural ... in the cortex and hippocampus, brain regions that ...
(Date:8/1/2014)... of marine mammals have officially named a species ... Sousa sahulensis , according to the Wildlife Conservation ... describing the newly named species is the culmination ... available historical records, physical descriptions, and genetic data ... ranging from the coast of West Africa to ...
Breaking Biology News(10 mins):Reptile Database surpasses 10,000 reptile species 2Reptile Database surpasses 10,000 reptile species 3Scientists name new species of cetacean: The Australian humpback dolphin 2Scientists name new species of cetacean: The Australian humpback dolphin 3
... African Americans are four to five times more likely to ... kidney failure have an increased risk of developing kidney failure, ... skewed risk between races. Previous studies identified variants in a ... gene creates a protein that is a component of HDL, ...
... innovative analysis of how social media can affect the ... by a scientist at Penn State University studying attitudes ... of biology, studied how users of Twitter -- a ... about a new vaccine. He then tracked how the ...
... Now computers are at it again, but this ... itself. An interdisciplinary team of scientists at Vanderbilt ... taken a major step toward this goal by demonstrating ... a biological system and derive the basic mathematical equations ...
Cached Biology News:Better ways to predict kidney disease risk for African Americans 2Better ways to predict kidney disease risk for African Americans 3Twitter data used to track vaccination rates and attitudes 2Twitter data used to track vaccination rates and attitudes 3Twitter data used to track vaccination rates and attitudes 4Robot biologist solves complex problem from scratch 2Robot biologist solves complex problem from scratch 3
... a platform for fully automated systems for ... easily be configured for applications ranging from ... allows closed loop screening, which means it ... generating, cherry picking and follow up studies ...
... Vivaclear centrifugal filters are disposable microfiltration devices for ... in the range 100 l to 500 l. ... accepting 2.2 ml centrifuge tubes. Product Features: ... size Low hold up volume ( ...
... The Caliper Prelude Workstation fully ... as solvent addition, extraction, sample transfer, ... on-line HPLC, and UV detection. The ... compliance with 21 CFR Part 11. ...
... Use in any standard fixed-angle microcentrifuge ... g.,Spin- centrifuge tube filters consist ... a centrifuge tube. They filter by ... HPLC sample preparation, removal of cells ...
Biology Products: