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Highly Sensitive an...
Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
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- Padhan, Narendra (författare)
- Uppsala universitet,Institutionen för immunologi, genetik och patologi,Rudbeck Lab
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(creator_code:org_t)
- 2018-09-19
- 2018
- Engelska.
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Ingår i: Journal of Visualized Experiments. - : JOURNAL OF VISUALIZED EXPERIMENTS. - 1940-087X. ; :139
- Relaterad länk:
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https://www.ncbi.nlm...
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visa fler...
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https://urn.kb.se/re...
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https://doi.org/10.3...
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Abstract
Ämnesord
Stäng
- Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pl), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Nyckelord
- Biochemistry
- Issue 139
- Capillary Isoelectric Focusing
- Signal Transduction
- ERK1/2
- Antibody
- Chemiluminescence
- Protein Isoforms
- Protein Phosphorylation
- Quantitation
- Ultrasensitive
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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