Detection of the low density lipoprotein (LDL) receptor on nitrocellulose paper with colloidal gold-LDL conjugates.
Gold-low density lipoprotein (LDL) conjugates were used to detect the LDL receptor on nitrocellulose paper. Solubilized rat liver membrane proteins were subjected to electrophoresis and electroblotted onto nitrocellulose paper. The receptor was then detected as a red band (within 10 min) by overlaying with the LDL conjugates. The coloration was prevented by unlabeled LDL, EDTA, and suramin but not by unlabeled HDL3. In the dot blot assay, detection with the colloidal gold-LDL conjugates was as sensitive as both the autoradiographic method with 125I-labeled LDL and the biotinylated LDL method; the estimated limit of detection by scanning densitometry was 1.6 femtomoles of receptor protein. When the coloration obtained with the colloidal gold-LDL conjugates was intensified by photochemical silver staining, down to 200 attomoles of the LDL receptor could be detected. In this assay, the EDTA-sensitive binding of colloidal gold-LDL to solubilized hepatic membrane proteins was 12 times higher for rats treated with 17 alpha-EE than for normal rats. The use of colloidal gold-LDL conjugates is therefore a very easy, safe, inexpensive, fast and sensitive method for the detection of the LDL receptor on nitrocellulose paper. Furthermore, with silver staining and scanning densitometry, the colloidal gold-LDL conjugates could be used in a dot blot assay to quantify tissue and cell LDL receptors down to attomolar levels.
Roach
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PD
Département de Biochimie, Université de Montréal, Canada.
Zollinger
M
M
Noël
S P
SP
D000818:Animals; D001710:Biotin; D003101:Collodion; D006046:Gold; D007457:Iodine Radioisotopes; D008099:Liver / Q000032:analysis; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D008722:Methods; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains; D011973:Receptors, LDL / Q000032:analysis*
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011973:Receptors, LDL; D001710:Biotin; D006046:Gold; D003101:Collodion