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- Table of Contents
Facts about DNA-(apurinic or apyrimidinic site) lyase 2.
Displays also double-stranded DNA 3'-5' exonuclease, 3'-phosphodiesterase activities. Shows robust 3'-5' exonuclease activity on 3'-recessed heteroduplex DNA and is able to remove mismatched nucleotides preferentially.
Human | |
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Gene Name: | APEX2 |
Uniprot: | Q9UBZ4 |
Entrez: | 27301 |
Belongs to: |
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DNA repair enzymes AP/ExoA family |
AP endonuclease 2; APE2; APEX nuclease (apurinic/apyrimidinic endonuclease) 2; APEX nuclease 2; APEXL2; Apurinic-apyrimidinic endonuclease 2; DNA-(apurinic or apyrimidinic site) lyase 2; DNA-apurinic or apyrimidinic site lyase 2; EC 3.1; EC 4.2.99.18; XTH2
Mass (kDA):
57.401 kDA
Human | |
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Location: | Xp11.21 |
Sequence: | X; NC_000023.11 (55000363..55009057) |
Highly expressed in brain and kidney. Weakly expressed in the fetal brain.
Nucleus. Cytoplasm. Mitochondrion. Together with PCNA, is redistributed in discrete nuclear foci in presence of oxidative DNA damaging agents.
Boster Bio APEX2 Marker employs the most advanced technology to image the APEX2 Fusion Protein. This antibody has been validated on various platforms and is proven to have high affinity and specificity. Additionally, Boster rewards the first reviewers of its products with product credits. This is an opportunity for scientists across the world to utilize the Boster biomarkers.
The detection of fusion proteins in cancer tissues is a challenge. Gross cytogenetic changes in different types of cancer are the most commonly used way to identify the presence of fusions. Chromosome banding analysis was initially limited to translocations and inversions with up to 3 megabytes in size. Additional challenges were posed by complex cytogenetics in specimens. Fortunately, recent advancements in imaging techniques have overcome these challenges.
Researchers have developed a multivalent nitrilotriacetic acid probe, dubbed P3NTA as a pioneering effort in the GFP fusion free FSEC method. The probe connects to the poly-histidine label that is fused to the membrane proteins that it is targeting. By using this method, fusion-free EM can detect fusion protein without the requirement to purify. The issue with this method is that the protein that is fusion-tagged may interact with other endogenous proteins.
There are numerous stains that detect Fusion proteins. InVision(tm) His-tag In-gel Stain is compatible with multiple downstream applications, including western blotting, Edman sequencing, and immunohistochemistry. The His-tag In-gel Stain can be used in combination with other stains, such as Coomassie and silver, to show bands of fusion proteins.
Multiple oncogenic fusions were discovered in cancer cells. Although these fusion proteins are only present in a tiny fraction of patients with malignancies they still require substantial investment to develop diagnostic tests or therapeutic substances. Multi-binding fusion proteins have multiple binding partners and are therefore more likely to be accessible for treatment. Thus, the development of an fusion protein-based treatment is crucial to the development of customized cancer therapies.
Two major kinds of fusion proteins are accessible. The HRP-Nb28 fusion protein is an extremely fluorescent protein that produces green light when excited by blue light. This protein also has the advantage of being stable under denaturing conditions, which allows researchers to use one genetic construct for multiple experiments. This makes it possible to detect fusion proteins within EM tissue without the use of chemicals or dyes.
The fusion protein OlZAC contains transmembrane and extracellular components. OlZAC is a pentamer containing transmembrane and extracellular elements. The analysis of OlZAC particles using a K3 direct detection camera was conducted. The results of this study suggested that OlZAC was an oligomer with monodispersed structures and high contrast after negative staining.
To determine the location of fusion protein, we utilized EM. In a single study, Sig1R–GFP-APEX2 was found to be located on both the cytosolic and NR sides of the membrane. The second transmembrane helix is believed to prevent Sig1R's native orientation. This question requires a high resolution approach.
A thin section of 70 nm was prepared for EM observations using an Leica EM UC7 ultramicrotome. It was counterstained with the uranyl acetate 8. The thin sections in series were then taken and examined using a Phillips CM120 STEAM electron microscope. The localization of fusion proteins EM requires a clear understanding of the mechanism for binding.
A fusion protein has significant effects on cell growth and activity, which is essential to the process of drug discovery. Therefore the fusion protein that has GFP is a good candidate to be used as an instrument for screening. GFP signals are located at the same site as Ab fusion proteins, making it easier to identify them. Further research is possible to evaluate the effectiveness of AD drugs by monitoring their distribution.
The use of fluorescent dyes, or fluorescent protein fusions in EM can be useful in determining the subcellular location of a specific protein. The problem with heterologous expression systems however they could have mistargeted or nonconserved signal sequencing. We propose using the pea Argenteum mutation as a model system. This method has the advantage of being simple to use and allows us to determine the exact place of fusion proteins.
A fusion protein that has a short linking sequence may be observed in neurons. However, it is unclear if this is an oligomer or one molecule. A recent study has revealed that long-linker Ab -GFP was not detected in the nucleus but was detected in other areas. The study demonstrated that the fluorescent protein was just one molecule, and that the results were in line with those from COS7 cells.
To identify Abmut–GFP, the gene that encodes the protein was transfected into COS7 cell lines. The cells were then stained with the 6E10 antibody. Abmut-GFP proteins are typically found in oligomers and not Tetramers in live cells. The fusion protein was not able to form any aggregates that could be detected in the cells, which was interesting. However Abmut-GFP proteins are observed in live cells.
The authors present a novel method of labeling that utilizes genetically-tagged proteins. It relies on the oxidation of diaminobenzidine in order to create silver/gold heavy atoms close to a protein. It can be utilized in conjunction with a variety electron microscopy techniques. The paper includes the results of three peer-reviewers. One of which is an active member of the Board of Reviewing Editors. Other reviewers included Yannick Schwab, Paul Verkade, and Anna Akhmanova. The Reviewing Editor has written a decision to assist the author in revising the submission.
Another approach is to combine APEX with nanobody-based detection systems. This combination is compatible both with 3D EM techniques as well as cryopreservation. This technique can be combined with immunogold labels to obtain higher signal-to-noise ratios. The resolution of images is comparable to or higher than that of immunogold labels.
Other methods to improve signal-to-noise using the APEX2 marker are the use of C-BERST in combination with multiplexing strategies. These strategies can be combined to improve signal-to noise ratio and proteomic mapping specificity. Background labeling could be due to factors like the distribution of amino acids that can be accessed from the surface, or the suitability of certain peptides for MS analysis. Two orthogonal dCas9s are another method that can increase the signal-to- noise ratio.
Another method involves the use of gold in addition to silver. This method requires an exact control of the time for development as well as silver enhancement and toning with gold. It is sensitive to actin-associated proteins as well as caveolar-proteins. It also produces an self-nucleated particles that are not real. It is important to conduct careful experiments to prevent this from occurring.
Combining APEX cytometry with other electron microscopic techniques can increase the signal-to-noise ratio by improving sensitivity. By combining APEX with other methods, scientists can detect endogenously expressed proteins in cells that have been altered by genetic editing. APEX-gold can also be used with freeze-substituted lowicryl sections and On-section labelling of Tokuyasu.
Coverslips-cultured cells were fixed with 4% PFA or 0.1% GA. After fixation cells were then infiltrated with 2.1 M sucrose at RT for 1 hr. The cells were then quickly frozen in liquid nitrogen to improve the signal-to-noise ration. The APEX2-positive cells will be more sensitive to biotin-labeling in the downstream steps.
SNR is directly related to the limit of detection. The detection limit is determined by the strength of the signal. A high linear dynamic range can assist in interpreting the SNR of an ELISA method. A high SNR allows for accurate analysis and identify large protein variances. These measurements can make a difference in the final analysis.
PMID: 11376153 by Tsuchimoto D., et al. Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen.
PMID: 16687656 by Burkovics P., et al. Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs.