Analysis of 1alpha,25-dihydroxyvitamin D3 receptor in cell extracts with low protein concentration.
An unacceptable loss of tritiated 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] to the wall of the reaction tubes constituted an obstacle when examining C3H/10T1/2 Cl 8 cells for 1,25-(OH)2D3 receptor. The loss of tracer in low protein cell extracts could be strongly reduced by incubating the cell extracts in polyethylene tubes and in the presence of inert peptides prepared by digestion of gluten proteins. When incubated in buffer the recovery of tracer increased from 3 to 45% by using polyethylene tubes instead of sodium-glass tubes. However, the presence of a sufficient amount of inert peptides in the buffer significantly increased the recovery of tracer to 89%. This procedure improved the saturation binding analysis of the 1,25-(OH)2D3 receptor in the C3H/10T1/2 Cl 8 cells using the hydroxylapatite assay.
Trydal
T
T
Department of Pediatrics, Haukeland Hospital, Norway.
Aksnes
L
L
Lillehaug
J R
JR
Aarskog
D
D
D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive; D002117:Calcitriol / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry*; D002460:Cell Line; D017886:Durapatite / Q000378:metabolism; D005983:Glutens / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism; D011095:Polyethylenes / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis; D018167:Receptors, Calcitriol / Q000032:analysis*; D012995:Solubility
D002457:Cell Extracts; D010446:Peptide Fragments; D011095:Polyethylenes; D011506:Proteins; D018167:Receptors, Calcitriol; D005983:Glutens; D017886:Durapatite; D002117:Calcitriol