How to understand antibody application（3）
We then went on to examine the interaction between proteins and nucleic molecules and the RNA immunisation continues to give you an idea of the application of antibodies
RNA immune precipitation
RNA immunological precipitation (RIP) is a method similar to ChiP, which is the interaction between qualitative and specific RNA sequences.
The main steps include: I) to form reversible protein-rna crosslinking with formaldehyde in living cells;
2) by using the specific antibody immuno-precipitation target protein;
(iii) reverse of crosslinking;
Iv) recovery and rt-pcr analysis of relevant RNA.
Experiment on electrophoresis migration
Electrophoresis mobility experiment (EMSAs) is used to study the affinity of DNA binding proteins to specific DNA sites.
In classic EMSAs, DNA fragments containing radioactive markers containing binding sites are co-incubated with target proteins.
Combination of affinity is according to the gel electrophoresis to determine the speed of the upward migration of DNA, due to the combination of DNA, protein molecular weight increased, resulting in slow migration in the gel electrophoresis.
In order to improve the specificity of the test, the specific antibody of target protein can be added in the reaction system.
If the antibody and target protein are combined to further slow the migration of the dna-protein complex in the gel electrophoresis, the superelectrophoresis experiment.
In-situ testing technique
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are two methods commonly used to detect tissue and protein expression and localization in situ.
The two methods are different in the sample preparation.
IHC is fixed on the tissue section, and the ICC is separated from the substrate of the cells in (for example, through blood cells centrifugal smear method, culture of cells can be grown into single cell layer) on the glass (figure 8).
Figure 8. Schematic diagram and representative results of the immunohistochemical techniques.
The mouse muscle ganglion was marked with antibody and laser confocal microscopy was used.
Green, tyrosine hydroxylase mark.
Blue, spontaneous fluorescence.
Fixed method
There are several common types of tissue or cell fixation used for IHC or ICC techniques, and the selection of fixed methods depends on the type of analysis.
The complete tissue samples used by immunohistochemical tests are usually used in formaldehyde, a semi-reversible cross-linking agent, which is derived from polyformaldehyde and can be further diluted to formalin.
Though research pointed out that in the formaldehyde fixed too much will have a negative impact on the sample, but also other reports that there is no negative impact, that is not fully fixed time will affect the formaldehyde crosslinking cell proteins.
The sample of the tissues of formaldehyde and even the entire animal sample was completely immersed in a formaldehyde solution with a concentration of 4 percent.
Fixed method
There are several common types of tissue or cell fixation used for IHC or ICC techniques, and the selection of fixed methods depends on the type of analysis.
The complete tissue samples used by immunohistochemical tests are usually used in formaldehyde, a semi-reversible cross-linking agent, which is derived from polyformaldehyde and can be further diluted to formalin.
Though research pointed out that in the formaldehyde fixed too much will have a negative impact on the sample, but also other reports that there is no negative impact, that is not fully fixed time will affect the formaldehyde crosslinking cell proteins.
The sample of the tissues of formaldehyde and even the entire animal sample was completely immersed in a formaldehyde solution with a concentration of 4 percent.
Table 3: immunohistochemical fixation methods.
The embedding method
The tissue must be buried in a matrix before slicing and staining.
Paraffin wax is commonly used to bury tissue.
Since paraffin is hydrophobic, the tissue must be dehydrated in a series of alcohols with a higher concentration gradient.
The penultimate step before perfusion is to immerse the tissue specimen in dimethylbenzene to dehydrate it completely.
In tissue perfusion after a short time after the paraffin, put them in the (usually in a small plastic box) 65% of paraffin bath, and then put into a mold, the final solidified into small pieces and by microtome section.
Paraffin embedding method is very popular because of its simplicity, low cost, persistent and high feasibility.
However, the paraffin embedding method also has the drawback, namely cannot cut very thin (less than 5 micron).
In order to be kept in high resolution optical microscope and electron microscope slices, choose plastic embedding is better, because you can cut into very thin tissue biopsies, and helps to keep organization form.
In addition, plastic can be successfully covered in very hard tissues (such as bone) and sliced.
Plastic bags are not commonly used, however, because they are more expensive than paraffin wax and interfere with tissue staining.
Previously frozen tissue can be buried with the best slice temperature complex (OCT), a water-soluble polyethylene glycol resin solution.
The background signal is low, so it is an ideal choice for the immunohistochemical staining of frozen tissue.
One of the obvious drawbacks of OCT embedding is that it becomes opaque when frozen and is difficult to locate when frozen.
Table 4: tissue embedding method in immunohistochemistry.
Table a reply
Although there are many advantages to using formalin fixation, it destroys the three-dimensional structure of epitope.
The table bit damage on the tissue slice can be reversed by the thermal induction epitope response (HIER).
The table bit response is generally combined with heating and acid/alkali solution;
Traditional methods often put the slices in a sodium citrate buffer solution with a pH of 6, but sometimes the high pH buffer is more effective in response to some antigenic epitopes.
Slice and buffer can be heated in very hot water bath, pressure cooker, pressure cooker or microwave, depending on the available equipment.
Marking/detection
The specimen can be stained in several ways.
The colorimetric method is commonly used by means of enzymatic antibody and colorimetric substrate.
This method is relatively simple, and the reaction is stable. The slice can be analyzed by means of a standard microscope.
However, endogenous enzyme activity or the non-specific binding of the endogenous agent to endogenous biotin can improve the background signal strength. Generally, one sample can only detect 1-2 target antigen.
In this regard, antibody detection by immunofluorescence or fluorescent dye markers is more advantageous because of simultaneous markers and detection of multiple target antigens.
However, it must be careful that the fluorescence dye of the coupling should be avoided in case the fluorescence quenched, because it is irreversible.
The histological method of immunisation can be used for high resolution analysis.
The samples were incubated with different sizes of gold particles, so that different antigens could be detected in a single sample.
These particles can be detected by high sensitivity and high resolution electron microscopy, which can accurately locate target antigen in cells and tissues.
Such staining is often used to verify subcellular localization or specific cell derived structures such as exosomes.
Table 5: immunohistochemical detection methods for antibodies.
Adjacent connection technology
It is always challenging to detect and characterize the interaction between two proteins.
Traditional methods, such as double-crossing experiments, are complex and present some limitations.
Ortho-connectivity testing (PLA) is a fast, sensitive and easy technology that can simultaneously detect and quantify protein interactions.
In addition, as a method of in-situ, it is possible to determine the intracellular position of the interaction in the pasted cell line and/or the frozen or paraffin embedding tissue.
The PLA can also quantify and detect individual endogenous proteins in natural conditions.
The PLA was first described by fredickson and others in 2002.
Since then, this technique has become one of the most widely used methods for analyzing the interaction of natural proteins without interference.
The PLA method is based on two antibodies labeled with oligonucleotides: these antibodies can bind to different table bits or two different proteins of the same protein.
Close to each other when antibodies (about 20-40 nm range), for example, when the two proteins to be analysed in interaction, antibody of oligonucleotide probe will work with the other two "joint oligonucleotide hybridization and combination, forming a continuous circular DNA structure.
At this point, the DNA polymerase will amplify the ring structure, and the ring structure will still be covalent with a PLA antibody.
The amplification of DNA molecules can be detected using standard fluorescence methods, which directly show the interaction between two pending proteins.
The PLA method is widely used: most commonly used to identify biomarkers in clinical Settings.
The PLA has also been used to study the role of specific proteins in biological processes, such as the development of cancer.
Recently, the PLA has been improved to directly detect post-translational modifications such as acetylation and phosphorylation.
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