Background[1-3]
Glyceraldehyde 3-phosphate dehydrogenase (rabbit-derived immunohistochemistry antibody) is a monoclonal antibody protein of glyceraldehyde 3-phosphate dehydrogenase prepared using rabbit as the host. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolysis process.
In addition to serving as a glycolytic enzyme in the cytoplasm, there is evidence that GAPDH in mammalian cells is involved in a variety of intracellular biochemical processes, including membrane fusion and microtubule formation. Microtubule bundling, phosphotransferase activation, RNA export from the nucleus, DNA replication and DNA repair. Some physiological factors, such as hypoxia and diabetes, can increase the expression of GAPDH in specific cells. GAPDH is present in almost all tissues and is continuously expressed at high levels. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme involved in glycolysis. It consists of four 30-40kDa subunits. GAPDH protein is expressed at high levels in almost all tissues and is widely used for Western blot The internal reference for protein standardization is a good internal reference antibody. As a housekeeping gene, the protein expression level of GAPDH is generally constant in the same cells or tissues. In the experiment, there may be inaccurate measurement of total protein concentration; or operational errors between samples caused when protein samples are loaded before electrophoresis; these errors need to be actually transferred to the membrane by measuring each sample. The content of GAPDH is used for calibration, so general western experiments require internal reference settings. The specific calibration method is to divide the measured target protein content of each sample by the GAPDH content of this sample to obtain the relative content of the target protein of each sample. Only then are sample-to-sample comparisons made.
Apply[4][5]
For the efficient preparation of glyceraldehyde 3-phosphate dehydrogenase and its application in prokaryotes and eukaryotes
Glyceraldehydes3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolysis reaction, providing energy for various life activities of organisms. This enzyme gene is a housekeeping gene and is generally expressed at high levels in tissues and cells. It can be used as an internal reference for RT-PCR, Western Blot and other experiments. In recent years, the new functions of glyceraldehyde 3-phosphate dehydrogenase have attracted more and more attention, such as activating its own transcription, initiating apoptosis, regulating the conversion between glycolysis and pentose phosphate pathways, etc. This study used the Escherichia coli prokaryotic expression system to prepare glyceraldehyde 3-phosphate dehydrogenase, constructed pET28b-GAPDH and TAT-pET28b-GAPDH recombinant vectors, and compared the effects of the addition of the protein transduction peptide TAT tag on glyceraldehyde 3-phosphate dehydrogenase. Effects of dehydrogenase expression. The results showed that the expression of glyceraldehyde 3-phosphate dehydrogenase was promoted through the addition of protein transducing peptide TAT tag, and the expressed protein had important enzymatic activity in vitro.
This study also studied the normal culture conditions and different conditions of prokaryotes (different strains of E. coli HB101, OP50, BL21, DH5α) and eukaryotes (yeast, Caenorhabditis elegans, rats, mice). Endogenous glyceraldehyde 3-phosphate dehydrogenation was identified under treatment conditions (different concentrations and treatment times of inducers, different concentrations and times of paraquat and sulfite). In addition, this study also compared the endogenous dehydrogenation of glyceraldehyde 3-phosphate through prokaryotic expression technology. Expression levels of sexual and exogenous glyceraldehyde 3-phosphate dehydrogenase. The results show that regardless of endogenous or exogenous glyceraldehyde 3-phosphate dehydrogenase, its expression remains relatively stable and is basically unaffected. Therefore, glyceraldehyde 3-phosphate dehydrogenase in prokaryotes and eukaryotes Can be used as a universal internal standard protein.
References
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[2]Identification of suitable reference genes for measurement of gene expression in human cervical tissues[J].Yuanming Shen,Yang Li,Feng Ye,Fenfen Wang,Weiguo Lu,Xing Xie.Analytical Biochemistry. 2010(2)
[3]A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1 TAT core domain[J].Yonghong Wu,Changhong Ren,Yan Gao,Bing Hou,Tingfang Chen ,Chenggang Zhang.Amino Acids.2010(3)
[4]Effect of naloxone on the induction of immediately early genes following oxygen-and glucose-deprivation in PC12 cells[J].Hsueh-Meei Huang,Jean-Yuan Yu,Hsio-Chung Ou, Kee Ching Jeng.Neuroscience Letters.2008(2)
[5] Zhang Xiao. Efficient preparation of glyceraldehyde 3-phosphate dehydrogenase and its application in prokaryotes and eukaryotes [D]. Northwest A&F University, 2012.
