Figure 1.
A model four span multipass membrane protein is shown in black with its C-terminus linked to the snorkel tag. L1 = linker 1, TMD = transmembrane domain, L2 = linker 2.
Figure 2.
Western blot analysis of the snorkel tag construct.
Plasmid constructs were transiently transfected into HEK293 cells, grown for 22h before analysis in western blot. A. Western blot with an anti-HA antibody. Lane 1; CD20 with a stop codon before the snorkel tag, lane 2; CD20 fused to the snorkel tag containing the HA epitope tag.
Figure 3.
Correlating membrane protein and snorkel tag expression.
Plasmid constructs were transiently transfected into HEK293 cells, grown for 22h before analysis in flow cytometry. A. Flow cytometry with CD20-snorkel cells. Transfected cells stained with anti-HA antibody (grey solid line) or with anti-CD20 (black dashed line); Grey dashed line (control); untransfected cells stained with an anti-HA antibody. B. same as A except with a CD20-stop construct. C CD20-snorkel dual stained with anti-HA (FITC labeled) and anti-CD20 (PE labeled). D. CD20-snorkel stained with CD20 antibody and the Alexa647 goat anti-mouse antibody (grey line), FRET measurement with the CD20-snorkel construct stained with anti-HA (Alexa488 labeled) and anti-CD20 and goat anti-mouse (Alexa647 labeled, black line). E same as D but plotted with both Alexa488 and 647 fluorescence for FRET pair.
Figure 4.
SNAP and CD24 snorkel constructs.
CD20 plasmid constructs were transiently transfected into HEK293 cells, grown for 22h before analysis in flow cytometry. A. Staining with a CD20 antibody, grey solid line; pSNKL-Q construct, black solid line; pSNKL-SNAP construct, dashed black line; non-transfected cells. B. same as A but stained with anti-HA antibody. C. same as A but with stained with the SNAP labeling reagent. D. CD20 in pSNKL-CD24. Black solid line; stained with CD20 antibody, grey solid line; stained with CD24 antibody, black dashed line; stained with HA antibody.
Figure 5.
Evaluation of the snorkel tag with a panel of membrane proteins.
Plasmid constructs based on pSNKL-Q with different membrane protein genes were transiently transfected into HEK293 cells, grown for 22h before analysis in flow cytometry. A. CXCR4, black dashed line; anti-CXCR4 antibody, grey dashed line; anti-HA antibody. B. same as A but with DARC and anti-DARC antibody. C through F were all stained with anti-HA antibodies either surface staining only (grey solid line), or with permeabilized cells (black solid line). C. CD20, D. Kir2.1, E. Kv1.3, F. Kir1.1.