Overview of the principle, classification and general operation steps of protein chips
Overview
Protein chips are also known as protein microarrays. The technology of protein chips was first proposed by Roger Ekin in the 1980s. It is a large array of protein molecules fixed to a carrier surface in a pre-arranged arrangement to form a microarray, according to the protein. The principle of intermolecular specific binding, the establishment of microfluidic biochemical analysis system to achieve accurate, rapid and large information detection of biological protein molecules, is a high-throughput, miniaturized and automated protein analysis technology. Simply Said, is an analysis technique that simultaneously detects hundreds or even thousands of target proteins/peptides in a single experiment.
Fundamental
Various proteins are sequentially fixed on various carriers such as titration plates, filters, and slides, and then proteins or other components labeled with specific fluorescence are applied to the chip, and the proteins on the chip are not rinsed. The components of the complementary combination are washed away, and then the fluorescence intensity of each point on the chip is measured by a fluorescence scanner or a laser confocal scanning technique, and the relationship between the protein and the protein is analyzed by the fluorescence intensity, thereby determining the various proteins. purpose.
The proteins immobilized on the chip can be: antigens, antibodies, small peptides, receptors and ligands, protein-DNA and protein-RNA complexes.
The antibody chip is the main type of protein chip. Its name is derived from the immunological point of view. It is widely used because of its huge potential application value in the detection of microbial infection. It is a branch of protein chip research which has a faster progress. . 2. The main detection methods are double antibody sandwich method and sample labeling method.
Take Raybiotech's antibody chip as an example:
Schematic diagram of the sandwich method:
The capture antibody is arrayed on a membrane or slide, incubated with the sample, and biotinylated antibody of the target protein is added. Finally, HRP-streptavidin or fluorescein-streptavidin is used to detect the chip signal.
Sample Marking Schematic:
The protein in the sample is labeled with biotin and then incubated with the capture antibody, and the control protein is added to the sample to monitor the entire reaction process, including biotin labeling and normalization. The protein bound to the chip was detected using HRP-streptavidin, and finally the signal was detected using chemical light or HiLyteTM Fluor 555-streptavidin.
General classification :
Classified by application:
1. Protein Detection Chips - A probe molecule with high affinity specificity (such as a monoclonal antibody) is immobilized on a substrate to identify a target protein/polypeptide in a complex biological sample. According to the detection method, different protein detection chips can be further divided into a normal phase protein detection chip and a reverse phase protein detection chip.
The normal phase protein detection chip first labels the proteins to be studied in the sample with different fluorescent labels, and then these samples are incubated on the antibody microarray, and then the fluorescence signals on the molecular points of each array are detected by a biochip scanner. .
The reversed-phase protein detection chip is a chip made by pulsing a small amount of tissue or a sample of a cell, and represents a protein of the whole cell under a certain state, and then is detected by a specific antibody.
2. Protein function chip----The natural protein spot is added to the substrate to understand which protein in the system can bind to a known protein for natural protein activity and molecular affinity study.
Classified by carrier
1 slide chip
(The picture shows a slide chip from Raybiotech, which has 16 sub-arrays, each of which can detect multiple indicators. It can be used for quantitative detection.)
2 membrane chip (using PVDF, or NC membrane as carrier)
(The picture shows a sub-array film chip)
The general steps of the protein chip :
(taking an antibody chip as an example)
The entire operation process includes:
1. Protein extraction from tissues or cells, body fluids;
2. Mark two samples with two different fluorescent molecules of Cy5 and Cy3;
3. Wash away excess marker molecules;
4. Incubation with the chip;
5. Scan the analysis results.
Signal detection and data processing of protein chips <br> The signal detection methods of mainstream protein chips are mainly grafted fluorescent detection systems for nucleic acid chips. The protein sample or the secondary antibody is labeled with a suitable fluorescent substance, and the fluorescence is excited by a confocal fluorescence scanner or a CCD fluorescence imager at a specific wavelength to obtain a signal for the reaction binding. In addition to such fluorescent label detection, another class of more attractive is the non-fluorescent label detection technology. The latter are represented by techniques such as gravity stress, surface nuclear magnetic resonance, and SPR (sllrfaee plasmon F esonance). In addition, it is based on the acoustic and optical principles of the Acoustic P late Mo de, elliptical optical detection. Their common feature is: high sensitivity, meeting the detection needs of trace proteins, but the disadvantage is that too high sensitivity often makes the distinction between garbage signals and effective signals difficult.
Demonstration picture:
An enlarged view of a subarray of protein chips .
The dot pattern seen in the subarray is the protein chip.
Strong and weak expression of internal signals. Can be detected by a variety of instruments
In terms of data processing, the current data processing of protein chips is mainly focused on how to quantify the protein concentration based on fluorescence intensity. Due to the huge difference in the content of various possible target molecules in biological samples and the diversity of protein interaction kinetics And complexity, how to divide false positive signals and positive weak signals have yet to be resolved.
Protein chips have the following advantages over traditional analytical methods :
1The amount of sample required is very small----micronization (10-100μl);
High-throughput parallel analysis of thousands of target proteins on a 2 protein chip----high throughput;
3 has a high signal to noise ratio, high accuracy, high sensitivity (monoclonal antibody);
4 fast, miniaturized and automated;
5 can be quantitatively tested by a standard curve;
6 Directly link DNA sequence information to protein products at the entire genome and proteome level.
The main application of protein chips
The trend of protein chips <br> The next step in the development of protein chips is the ability to analyze the entire proteome, which means that at least 5,000 to 10,000 proteins are arranged on a single chip.
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