How to understand antibody application（2）

We go on to a protein test and a purified immune PCR

Immune PCR

Immunopcr (i-pcr) combines the sensitivity of PCR amplification and specificity based on antibody determination, so this technique increases detection sensitivity.

In general, the detection limit of the ELISA method can be increased from 100 to 10,000 times in different applications.

Ⅰ - PCR was first described in 1992 by sano et al.

Despite the potential of the technology, the use of the i-pcr method in diagnostics and biomedical applications has been limited by several issues for several years, including the duration of the test, resulting in an increase in the error rate.

In addition, one of the main drawbacks is that antibodies are difficult to combine with oligonucleotides.

Several solutions have been suggested, such as rapid and efficient combination of methods, such as Innova's Biosciences company Thunder - Link ® antibody - oligonucleotide combined system).

In addition, the introduction of the multiple and high throughput technology greatly improved the I - polymerase chain reaction (PCR), it has been used as a routine diagnostic tests of some bacteria and virus pathogens, and used for detection of tumor markers and food contamination.

Quantitative PCR combined with i-pcr (q-pcr) can be used to quantify low-abundance biomarkers in complex biological samples, which is difficult to quantify by traditional immunological methods.

• Proteins molecularly imprinted

Immunoblotting

Protein imprinting is a kind of protein that separates proteins through gel electrophoresis and transfers the protein to the adsorption membrane.

Once the protein is printed, the protein can be detected by specific marker antibodies.

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS - PAGE) can be used to detect the protein that has been chemically modified by SDS.

However, certain antibodies do not recognize their epitopes on the protein molecules of a denatured protein, so the SDS polyacrylamide gel electrophoresis is required.

Protein imprinting can be done by wet, semi-dry, or dry.

In the wet process, the gel is sandwiched between the imprinted film and various filtration devices, and then the entire device is buried in a electrode slot filled with triglycine transfer buffers.

In semi-dry, there is only a small amount of buffer around the gel in the middle and then closed between the electrodes.

Finally, there is no need for buffer fluid in the dry system, using pre-assembled devices with electrodes, buffer and membrane.

Although the dry method transmembrane is relatively fast, it needs to fit the interlayer parts of each part well, while the wet process film has very few problems, so as to keep the consistency of the experimental results as far as possible.

In addition, if the subsurface of the semi-dry process is too long, the protein of the smaller molecular weight may be turned from the membrane.

After the membrane is transferred, the membrane should be closed to reduce the non-specific protein binding, and then use an anti-incubation membrane to study the target protein of interest.

Monoclonal antibodies usually have higher specificity;

However, many monoclonal antibodies are produced in a specific peptide segment rather than a natural protein, so it may not be possible to detect the natural proteins that PAGE has separated.

Polyclonal antibodies may be used, but higher background values are easily formed.

Because an anti-resistance is usually unmarked, it requires a two-reactance that can be combined in an anti-fc segment to be used for final testing.

They are usually labeled with enzymes.

The marks of the enzyme can be imaged by chemiluminescent substrate and radiographic imaging.

Proteins that are detected and tagged by antibodies appear in dark areas of the image.

Figure 6. Immune imprinting.

A. Flow diagram.

B. Representative results.

Dot Blot

The dot imprinting is similar to the immune imprinting method, which detects the target protein on the membrane.

But the protein detected in the dot imprinting method is not separated by electrophoresis.

Instead, samples containing the target protein were directly "dots" to the membrane.

This is not a quantitative method, but it can directly detect the presence of a particular protein.

For example, the spotted imprinting method can be used to detect certain protein localization in the cell lysis products of sucrose gradient separation.

• To detect the interaction between proteins - nucleic acid molecules

  
  

Chromatin immunity precipitation

The chromatin immunological precipitation (CHIP) was first developed in the 1980s in the study of the interaction between RNA polymerase II and target genes.

In the process, cells are fixed in formaldehyde or similar fixers to make cellular DNA and related protein molecules cross.

Fixed cells via ultrasonic processing, then make DNA - protein complexes in the cells rupture is 100-300 bp fragments, and then use bind to the DNA of specific proteins (such as proteins and transcription factors) antibodies, in accordance with the relevant standard method for precipitation of DNA fragments.

Finally, the PCR analysis was used to combine the DNA fragments of the protein with the separation of DNA - protein complex.

At the same time, protein and DNA are deposited together, which can be identified by mass spectrometry.

Ideally, the standard CHIP method is used to verify a protein and a assume that the interaction between target genes, because according to assume that the target gene sequence design specific PCR primers (figure 7).

Figure 7. Process schematic diagram of chromatin immunity precipitation.

There is a limitation in CHIP method that the crosslinking step may destroy the protein molecular structure, thus impeding the antibody and its binding, affecting subsequent immune precipitation.

At this point, you can try the CHIP method without the crosslinking step;

This process is called the n-chip immune precipitation method.

Although crosslinking has been used to improve antigen recognition, it is generally applicable only to target proteins that are strongly associated with DNA.

ChIP - on - ChIP

Recently, chips have been modified to be used for high-throughput analysis.

For example, a ChIP - on-chip combination of ChIP and microarray technology can be applied to a whole-genome screening of a fluorescent tagged sequence.

In these tests, using different DNA DNA fluorescent tags to precipitate group and the control group (usually without precipitation total input), and then use each group of DNA and DNA microarray chip, and even the whole genome small oligonucleotide hybridization.

By standardizing the analysis techniques, we can obtain detailed information about the DNA binding sites of CHIP samples relative to the DNA control group.

Chromatin immunity co-precipitation sequencing

By combining chromatin precipitation technology with modern high-throughput sequencing technology, it is beneficial to identify the target gene sequences unknown before.

Because high-throughput sequencing can identify and provide a large number of genome information, chromatin immune-co-precipitation sequencing can be applied to the chromatin immunological precipitation detection of the whole genome.

This method is a very powerful tool for identifying the binding sites of genomic DNA and proteins such as transcription factors to reveal biological processes and gene regulation.

Recently, a method called endogenous protein rapid immunomass spectrometry (RIME) based on mass spectrometry (RIME) has been proposed to combine with chip-seq.

This combination method provides two types of information for the target protein's cis-antigroup and interaction groups.

Cross-linking immune precipitation

Crosslinked immunoprecipitation (CLIP) is a method developed by maille et al. in 2003.

They studied the interaction between the splicing factor NOVA and the neuron specific RNA binding protein (RBP).

Similar to chromatin immuno-precipitation techniques used to analyze the interaction of dna-protein, crosslinked immunoprecipitation technology can be used to analyze the interaction of rna-protein.

The method of chromatin immunity coprecipitation was similar to chromatin immunisation.

However, because the target nucleic acid is different, there are also several obvious differences.

In the sequence of chromatin immune coprecipitation, the recommended crosslinking agent is uv irradiation.

Since transcriptional RNA is generally shorter, it does not need to be treated with ultrasound, and the cells can lyse in standard buffer.

However, the RNase inhibitor must be added to all pyrolysis products to prevent the degradation of target RNA.

CLIP can analyze RNA - protein interactions and map RNA binding sites of the entire genome.

In particular, the HITS - CLIP has been widely used to draw several splicing factors, such as PTB, FOX2, and Argonaute protein-rna interaction sites.

However, the HITS - CLIP shows some of the shortcomings related to the precise measurement of crosslinking efficiency and RBP binding sites.

In order to overcome these problems, in 2010 ha, and others developed light activation - ribose nucleoside increased crosslinking and precipitation (PAR - CLIP), in 2011 konig proposes individual nucleotides solutions such as CLIP (iCLIP), provides the RBP binding sites in single nucleotide level solutions.

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