Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents.
We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine-HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.
Caughey
D J
DJ
Amgen Inc., Thousand Oaks, CA 91320-1789, USA. dcaughey@amgen.com
Narhi
L O
LO
Kita
Y
Y
Meng
S Y
SY
Wen
D
D
Chen
W
W
Ratzkin
B J
BJ
Fujimoto
J
J
Iwahara
T
T
Yamamoto
T
T
Arakawa
T
T
D000077548:Anaplastic Lymphoma Kinase; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D020794:Receptor Protein-Tyrosine Kinases; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012997:Solvents
D000906:Antibodies; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012997:Solvents; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases