Fig. 1

HCAb2 preferentially bound to the surface of MDA-MB-231 cells. a. Alignment of amino acid sequences of HCAb1 and HCAb2 revealing mutations (*) in comparison to their respective germline VDJ sequences. HCAb1 and HCAb2 nucleotide sequences were analyzed using IMGT/V-QUEST program to determine VDJ gene segments of the antibodies as well as mutations in complementarity determining regions (shaded) and framework regions. b. Immunoblot depicting differences in monomeric molecular weights of 8 different heavy chain antibodies. Purified heavy chain antibodies (~300 ng) were run on a reducing SDS-PAGE gel, transferred to a nitrocellulose membrane and detected using anti-mouse IgG antibody. c. Flow cytometry screening of MCF10A, MCF7 and MDA-MB-231 cells using HCAb1 and HCAb2. HCAb1 (green peak), HCAb2 (yellow peak), isotype control (red peak) and unstained (blue peak). d-o Immunofluorescence analysis of HMEC, MCF7 and MDA-MB-231 cells using HCAb1 and HCAb2. d-i Non-permeabilized cells were stained with HCAb1 (d-f) and HCAb2 (g-i) to determine cell surface staining. j-o Permeabilized cells were stained with HCAb1 (j-l) and HCAb2 (m-o) to determine intracellular staining. Scale bar represents 10 μm