Bosterbio
Anti-Dopamine D2 Receptor Antibody
PA2234
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This antibody was tested in a study to investigate the feasibility of measuring D2R levels in rat brain via WB. We attempted to validate this antibody in various positive and negative controls, however we did not observe the expected changes in band size among our different samples and the blot was polluted with numerous non-specific bands.
Western Blot
1.) Human Sy5y cells, 2.) Human SY5Y differentiated for DAergic phenotype, 3.) HEK cells, 4.) HEK-cells transfected to overexpress D2R, 5.) HEK cells transfected to overexpress D1, 6.) Rat midbrain homogenate, 7.) Rat cerebellar homogenate, 8.) Recombinant human D2R protein, 9.) Rat primary neurons 8 DIV
0.5ug/ml overnight at 4 deg. celcius in TBS-T with 1x NAP
1x NAP in TBS-T 1 hr RT
Anti-rabbit HRP (1:3000)/ Anti-biotin HRP (1:3000- 6000) for 1 hour at RT
None
Pierce chemiluminescence kit (#PI34096)
We observed bands at 20, 40, 50 60, and 100 KD, (expected band size 50 KD) in all tissues/cells tested. The most prominent band appeared at 20 Kd. We saw no different in band size between our positive and negative controls. + controls human lanes: 1 (DA neuron differentiated SY5Y cells), 4 (HEK cells transfected to overexpress D2R)- control human: lanes 2 (undifferentiated SY5Y), 3 (HEK -parental line), 5 (HEK cells transfected to overexpress D1R)+ control rat: lane 6 (rat midbrain homogenate)-control rat: lane 7 (rat cerebellum homogenate)These controls have been previously been used to sucessfully validate D2R detection by other antibodies (sc-5303)
N/A
Ease of use, bands were at least visable
Non-specific detection
This antibody is unacceptable for detection of D2R in human or rat tissue.