Fig 1: Validation of IDH1 antibody using purified recombinant protein in multicolor and chemiluminescent Western blotting. Multicolor and chemiluminescent Western blottings were performed using 10% Bis-Tris SDS-polyacrylamide gel and MOPS buffer system to validate the IDH1 antibody using a purified recombinant IDH1 protein (0.16 µg) containing a c-Myc tag in addition to HEK293T and HeLa whole-cell lysates. A, c-Myc protein tag present on the purified IDH1 recombinant protein is detected in the 700-nm channel (red) at 50 kDa via mouse anti-c-Myc antibody (ab32;1 µg/ml) using IRDye 680RD goat anti-mouse IgG (H + L) for detection. Some overspill of the recombinant protein into neighboring lanes is observed (white box). B, IDH1 recombinant protein and endogenous IDH1 protein, present in HEK293T and HeLa, is detected in the 800-nm channel (green) at 55 and 50 kDa, respectively, using rabbit anti-IDH1 antibody (ab172964; 1.2 µg/ml) and IRDye 800CW goat anti-mouse IgG (H + L) for detection. C, when both 700- and 800-nm channels are displayed, the signal from ab32 and ab172964 overlaps at 50 kDa, identifying the c-Myc–tagged IDH1 protein. No overlap is seen for the endogenous IDH1 present in HEK293T and HeLa whole-cell lysates. A–C, lysates loaded per lane are as follows: 20 µg of blocking buffer: Odyssey blocking buffer (TBS); imager: Odyssey® CLx; resolution: 169 µm; intensity: auto mode. ChameleonTM Duo pre-stained protein ladder for accurate sizing of protein bands. D, single blot was split into two halves (green line) to be incubated with either rabbit anti-IDH1 antibody (ab172964; 0.115 µg/ml) or the corresponding rabbit monoclonal IgG isotype control (ab172730; 0.166 µg/ml) to detect the endogenous IDH1 protein present in HeLa and HEK293T as well IDH1 recombinant protein. Both halves were incubated with HRP-conjugated goat anti-mouse IgG (H + L). E, single blot was split into two halves (green line) to be incubated with either mouse anti-c-Myc antibody (ab32; 1 µg/ml) or the corresponding mouse monoclonal IgG1 isotype control (ab18443; 1 µg/ml) to detect c-Myc protein tag present on the purified IDH1 recombinant protein but absent in HEK293T and HeLa whole-cell lysates. Both halves were incubated with HRP-conjugated goat anti-rabbit IgG (H + L). Blots were detected with WesternSure® PREMIUM chemiluminescent substrate (LI-COR 926–95000) and imaged on an Odyssey® Fc with the following resolution: 125 µm and exposure of 2 min. Lysate loaded per lane: 20 µg; protein ladder: WesternSure® pre-stained chemiluminescent protein ladder (LI-COR 926-980000); blocking buffer: intercept blocking buffer (TBS); intercept T20 (TBS) antibody diluent.