Human Choline Acetyltransferase/ChAT Antibody

Detection of Human Choline Acetyltransferase/ChAT by Dot Blot

Detection and characterization of extracellular ChAT protein in human plasma and CSF.(a) Dot-blot analysis of nine different pooled CSF samples using two different antibodies. (b) The relative amount of ChAT in plasma or CSF, compared to brain homogenates (BH) of AD or control. At 16-fold dilutions, the immunosignals are much stronger in plasma and CSF compared to BH. This demonstrates the relative abundance of the ChAT protein in extracellular fluids. (c) Western-blot characterization of molecular form of ChAT in plasma, CSF and BH. The major detected protein band (*) in the BHs corresponds to a ChAT protein with a Mw of ∼65 kDa. In addition, there are several detected heavier molecular forms of ChAT in the CSF. All samples were loaded on one gel. Note also that the amount of the ∼65 kDa ChAT was so high in plasma that it distorted the gel downward. (d–g) Combinatorial sandwich ELISA results for identification of extracellular ChAT using three different antibody pairs for recombinant human ChAT (rhChAT), and a pooled plasma sample (g) calibrated for ChAT protein concentration using the rhChAT and ELISA setup in (f). (h) Further characterization of the molecular forms of CSF ChAT in consecutive fractions of pooled AD CSF samples separated by sucrose-density gradient technique, and the subsequent quantification of ChAT by sandwich ELISA. The graph represents the average of nine different pooled CSF. This independent analysis provides an identical pattern of molecular forms detected by Western analysis. Due to lack of prior reports, we used analogous terminology, which is used for the counteracting cholinergic enzymes, AChE and BuChE, that is, Gn, where n denotes the number of globular subunits in each detected molecular form of ChAT in CSF. The molecular weights are calculated based on the known Mw of two internal standard proteins. In all dot-blot analyses, 2 µL of each sample (neat or diluted) was used. In the Western blot analysis, each lane was loaded with 15 µL of a mixture, containing 10 µL of sample (neat or diluted) and 5 µL of a 6x concentrated reducing Laemmli loading buffer. All ChAT protein quantifications were done with the ELISA antibody pair’s combination in (g). Anti-ChAT antibodies: RP = rabbit polyclonal antibody (Ab), BGP = biotin-labeled goat polyclonal Ab, MAB = mouse monoclonal Ab. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0065936), licensed under a CC-BY license. Not internally tested by R&D Systems.