Rapid isolation of phage displayed antibodies to beta-actin eluted from two-dimensional electrophoresis gel.
We describe a simple and efficient procedure which can be used to prepare antibodies to proteins extracted by two-dimensional gel electrophoresis (2-DE), using beta-actin as a model. Protein was electroeluted from a stained gel, biotinylated and used for selection of phage from a semisynthetic phage antibody library. After four rounds of selection using 50 ng beta-actin per cycle, approximately 8 X 10(3) phage were recovered. Antibody fragments were prepared from 21 randomly picked clones. Six of eighteen (6/18) antibody-positive clones produced antibody fragments reacting against beta-actin in an enzyme linked immunosorbent assay (ELISA). Sequencing of the HC-CDR3-region showed that all six clones were independent isolates, suggesting that a large number of independent phage antibody reactivities were generated.
Radiumhemmets Research Laboratory, Cancer Center Karolinska, Karolinska Institute and Hospital, Stockholm, Sweden.
J N
JN
Zhou
S
S
Linder
B
B
Franzén
G
G
Auer
D F
DF
Hochstrasser
M A
MA
Persson
D000199:Actins / Q000276:immunology*; D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification*; D001435:Bacteriophages; D001710:Biotin; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005782:Gels; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000032:analysis; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D013997:Time Factors
D000199:Actins; D000906:Antibodies; D005782:Gels; D007135:Immunoglobulin Variable Region; D001710:Biotin