Schema and validation for isolation and identification of autoreactive CLL surface proteins. Panel A alone depicts a schematic representation of an idealized antigen-identification process. Primary CLL cells were surface labeled with biotin. Lysates were loaded onto streptavidin columns, and biotinylated membrane proteins were collected. Cell surface proteome aliquots were run on two identical sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. One gel was transferred to nitrocellulose and probed with autologous serum at a dilution of 1:100, probed with anti-human IgG-HRP, washed, and developed by autoradiography. Any autoreactive bands were isolated from the coomassie stained gel and subjected to mass spectroscopic protein identification. (B) A coomassie stained gel shows relative protein recovery at each step in the isolation of membrane proteins from freshly purified primary CLL B cells. (C) Immunoblot analysis of cytosolic proteins β-actin and α-tubulin as well as membrane proteins CD20 and CD52 demonstrates enrichment of membrane protein isolation from primary CLL cells. (D) MEC1 CLL cells containing a doxycycline-inducible CD37 construct were utilized to demonstrate the fidelity of cellular membrane isolation. (E) Membrane fractions from primary CLL B cells isolated from two different patients on two different days to ensure repeatable enrichment of membrane proteins.