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:12/24/2014)... Texas , Dec. 23, 2014 ... company focused on gynecologic disease, announced today that ... Schuler , Birchview Fund LLC and several Vermillion ... of unregistered shares of Vermillion,s common stock and ... stock in a private placement.  ...
(Date:12/24/2014)... , Dec. 23, 2014  Rock Creek Pharmaceuticals, Inc., ... has filed a Clinical Trial Application (CTA) with the ... Regulatory Agency (MHRA) seeking regulatory approval to initiate clinical ... Anatabine Citrate. Contingent on the Company receiving ... Phase I trial to assess the safety, tolerability and ...
(Date:12/22/2014)... Md. , Dec. 22, 2014 /PRNewswire/ ... SYN), a developer of pathogen-specific therapies for ... on protecting the microbiome, today announced positive ... Phase 1a clinical trial of SYN-004, the ... prevention of Clostridium difficile (C. difficile) ...
(Date:12/22/2014)... DIEGO , Dec. 22, 2014 GenomeDx ... the Decipher Prostate Cancer Classifier, a genomic test for ... in node-negative, high-risk men managed by radical prostatectomy without ... radical prostatectomy is relatively uncommon, using tumor genomics to ... of metastatic disease is an important advance. The study ...
Breaking Biology Technology:Vermillion Announces Equity Financing of up to $18.9 Million; Suspends ATM Program 2Vermillion Announces Equity Financing of up to $18.9 Million; Suspends ATM Program 3Vermillion Announces Equity Financing of up to $18.9 Million; Suspends ATM Program 4Vermillion Announces Equity Financing of up to $18.9 Million; Suspends ATM Program 5Rock Creek Pharmaceuticals Files Clinical Trial Application in the UK for Clinical Trials of Anatabine Citrate Formulations 2Rock Creek Pharmaceuticals Files Clinical Trial Application in the UK for Clinical Trials of Anatabine Citrate Formulations 3Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 2Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 3Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 4Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 5Study Published in European Urology Shows that the Decipher Prostate Cancer Classifier is Predictive of Rapid Metastasis in High-Risk Men who have had Prostate Surgery 2Study Published in European Urology Shows that the Decipher Prostate Cancer Classifier is Predictive of Rapid Metastasis in High-Risk Men who have had Prostate Surgery 3
... BALTIMORE, June 14, 2011 Arcion Therapeutics , ... therapies for chronic pain, today announced that data from ... of pain associated with painful diabetic neuropathy (PDN) will ... medical meetings.  The first poster will be presented at ...
... PAREXEL International Corporation (NASDAQ: PRXL ) announced ... Healthcare Conference in Boston, MA.  James Winschel, Sr. Vice President ... PAREXEL at 2:00 p.m. ET on Wednesday, June 22, 2011. ... available through the "Investors" section of PAREXEL,s website ...
... 2011 /PRNewswire-Asia/ -- TWi Biotechnology Inc. announced today ... the total 240 patients for the Phase IIb ... Type II Diabetes. The randomized, double-blind, placebo-controlled, dose-ranging, ... glycated hemoglglobin A1c (HbA1c) lowering effects of AC-201 ...
Cached Biology Technology:Arcion Therapeutics to Present ARC-4558 Phase 2b Data for the Treatment of Pain Associated With Painful Diabetic Neuropathy at Upcoming Medical Meetings 2Arcion Therapeutics to Present ARC-4558 Phase 2b Data for the Treatment of Pain Associated With Painful Diabetic Neuropathy at Upcoming Medical Meetings 3PAREXEL International to Present at Wells Fargo Healthcare Conference 2Positive Results from Phase IIa Study Pave Way for Phase IIb Trial of AC-201 for the Treatment of Type II Diabetes 2
(Date:12/22/2014)... 2014  NXT-ID, Inc. (NASDAQ: NXTD ... company focused on the growing mobile commerce market, announces ... Apollo Robbins for the 2015 International CES ... Apollo Robbins will be at the ... demonstrating some of his famous theft techniques to visiting ...
(Date:12/19/2014)... MOUNTAIN VIEW, Calif. , Dec. 18, 2014 /PRNewswire/ ... the publication of a study that pinpoints fine-scale differences ... the United States . Since immigrants ... the United States has served as ... study illuminates how American history and the ongoing mixing ...
(Date:12/17/2014)... Valencell, a leader in performance biometric data ... industry leaders such as Intel, Jabra, Atlas, and Scosche ... products. These products will be showcased at CES 2015, ... . "Our partners choose PerformTek ... – with the clinical data to back it up," ...
Breaking Biology News(10 mins):NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 2NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 3NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 423andMe Study Sketches Genetic Portrait of the United States 223andMe Study Sketches Genetic Portrait of the United States 323andMe Study Sketches Genetic Portrait of the United States 4Valencell PerformTek Biometrics Power the Most Accurate Wearables at CES 2015 2
... Columbia have been able to pinpoint more of the ... valuable compounds to the roasting process. Lead author ... the prevailing antioxidants present in dark roasted coffee brew ... high temperatures. Their findings will appear in ...
... Zaragoza (UNIZAR) has studied precipitation trends in Spain,s 10 ... results show that precipitation has declined overall between the ... the rainy season. The rains are heavier in October ... 1946, the average precipitation falling on Spanish hydrological basins ...
... a serious environmental, economic and social problem worldwide as ... ecosystem functions, such as nutrient cycling. Despite substantial research, ... new habitats over native plants that technically should have ... is that these plants are more abundant in introduced ...
Cached Biology News:Roasting coffee beans a dark brown produces valued antioxidants: UBC food scientists 2Rain in Spain is on the decline 2Home and away: How do invasive plant species dominate native species? 2
Mouse monoclonal antibody raised against a partial recombinant PDE2A. NCBI Entrez Gene ID = PDE2A...
... array sample elements. , , ,Includes: , , ... in PBS (10X): 50 ml , Streptavidin ... 8 packs , 10% Tween-20: 2 x 10 ... x 25 ml , SuperSignal West Pico Stable Peroxide: ...
... polyclonal antibody raised against a ... Immunogen: XAGE2 (NP_570133, ... partial recombinant protein with GST ... Number: NM_130777 ...
...
Biology Products: