Sheep Alpha-Granular membrane protein 140 ELISA Kit

Background High resolution melting (HRM) analysis is one of the newer, reliable, and sensitive genotyping techniques, which offers considerable time and cost savings. P-selectin is an adhesion molecule that has a role in the initial phases of leukocyte adhesion to stimulated platelets and endothelial cells in inflammation. Multiple polymorphisms in P-selectin gene (SELP) that affect the protein sequence have been described. The aim of this study was to design, optimize, and validate a simple and rapid in-house HRM-based method for genotyping the NM_003005.3:c.992G>A (c.992G>A), NM_003005.3:c.1918G>T (c.1918G>T), and NM_003005.3:c.2266A>C (c.2266A>C) SELP polymorphisms. Methods Initial genotyping of three SELP polymorphisms was performed by applying polymerase chain reaction (PCR) with sequence-specific primers (SSP), which was used as a reference method for determination of analytical sensitivity. PCR-HRM was performed with primers for c.2266A>C reported in the literature. Primers for the remaining two polymorphisms were designed using Primer-BLAST. Precision testing was performed using three samples with different genotypes. For accuracy, analytical sensitivity and specificity testing, 20 wild type, 10 heterozygous, and 10 homozygous samples were chosen per polymorphism. Results were expressed as percentage of concordance with the acceptability criterion >= 95%. Results Agreement of results was 100% for all validation parameters except for analytical sensitivity for c.1918G>T and c.2266A>C, with agreement of 90%. Repeated analysis using both methods revealed an error in initial genotyping and correct genotyping by PCR-HRM, which was confirmed by Sanger sequencing. Conclusion The validation confirmed PCR-HRM as a precise, accurate, and specific method for genotyping the c.992G>A, c.1918G>T, and c.2266A>C SELP polymorphisms.

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome -positive (Ph(+)) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.