Synthesis of a yohimbine-agarose matrix useful for large-scale and micropurification of multiple alpha 2-receptor subtypes.
We have provided a detailed protocol for the synthesis of a yohimbine-agarose matrix that has been shown to be effective for isolation of the alpha 2A-adrenergic receptor from human platelet and purification of the alpha 2A-adrenergic receptor to apparent homogeneity from porcine brain cortex using chromatography on only two sequential yohimbine-agarose columns. In addition, this affinity matrix also interacts with alpha 2 receptors of the alpha 2B subtype extracted from cultured NG108-15 cells. Finally, this affinity matrix has proven useful for monitoring posttranslational modifications of the receptor in digitonin extracts of metabolically labeled cells. Thus, this affinity matrix can be exploited for the purification of multiple alpha 2-adrenergic receptor subtypes on both a macro- and microscale and should be of value to any laboratory exploring the molecular basis for alpha 2-adrenergic functions.
Domino
S E
SE
Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor 48109.
Repaske
M G
MG
Bonner
C A
CA
Kennedy
M E
ME
Wilson
A L
AL
Brandon
S
S
Limbird
L E
LE
D000316:Adrenergic alpha-Agonists / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D004072:Digitonin; D006801:Humans; D007202:Indicators and Reagents; D011869:Radioligand Assay / Q000379:methods; D011942:Receptors, Adrenergic, alpha / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D014316:Tritium; D015016:Yohimbine / Q000031:analogs & derivatives* / Q000097:blood / Q000138:chemical synthesis
D000316:Adrenergic alpha-Agonists; D007202:Indicators and Reagents; D011942:Receptors, Adrenergic, alpha; C077867:yohimbine-agarose matrix; D014316:Tritium; D015016:Yohimbine; D012685:Sepharose; D004072:Digitonin