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Western blot (or immunoblotting) is used for the detection of
specific proteins. After SDS-PAGE, the gel is transferred to a nitrocellulose
membrane or other solid support. Proteins are detected by treatment with specific
antibodies and revealed using labeled anti-Ig antibodies.
Chimeric ETA(dII)/E7 DNA construct and characterization of
E7 protein expression. A, schematic diagram showing the constructs of full-length
ETA and the chimeric ETA(dII)/E7 gene. The DNA fragment encoding ETA(dII) (aa
247–416) is depicted in the . The fragment encoding HPV-16 E7 (aa 1–96)
is depicted in the . B, Western blot analysis to characterize the expression
of E7/GFP protein in cells transfected with E7/GFP or ETA(dII)/E7/GFP DNA. Lane
1, lysates from cells transfected with E7/GFP DNA; Lane 2, lysates from cells
transfected with ETA(dII)/E7/GFP DNA; Lane 3, concentrated culture medium from
cells transfected with E7/GFP DNA; Lane 4, concentrated culture medium from
cells transfected with ETA(dII)/E7/GFP DNA; Lane 5, lysates from nontransfected
293 Db, Kb cells as a negative control. Note: lysates from E7/GFP DNA-transfected
293 Db, Kb cells revealed a protein band with a size of approximately Mr 30,000
corresponding to E7/GFP protein in Lane 1, as indicated by the short arrow.
Meanwhile, lysates from ETA(dII)/E7/GFP DNA-transfected 293 Db, Kb cells generated
a protein band with a size of approximately Mr 56,000 corresponding to ETA(dII)/E7/GFP
protein in Lane 2, as indicated by the long arrow. E7/GFP DNA-transfected cells
exhibited levels of protein expression comparable with that of ETA(dII)/E7/GFP
DNA-transfected cells.(Cancer Immunotherapy Using a DNA Vaccine Encoding the Translocation Domain of a Bacterial
Toxin Linked to a Tumor Antigen)
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