b'Without enhancer SuperSignal Western Blot EnhancerCDK 50.625 1.25 2.5 5 10 g 0.625 1.25 2.5 5 10 g Figure 53. SuperSignal Western Blot Enhancer improves the lower detection limit for chemiluminescent substrates. Cell lysates were GSK3 separated by electrophoresis, transferred to nitrocellulose, and western blotting was performed using the conventional method (left) or using the SuperSignal Western Blot Enhancer protocol (right). 0.625 1.25 2.5 5 10 g 0.625 1.25 2.5 5 10 gTop panel Bottom panelBlocking buffer 5% milk in Tris-buffered saline with 0.05% Tween 20 surfactant Thermo Scientific Blocker BLOTTO in TBSPrimary antibody Mouse anti-CDK5 at 1 g/mL Rabbit anti-GSK3 at 1 g/mLSecondary antibody HRP-conjugated goat antimouse IgG at 0.1 g/mL HRP-conjugated goat antrabbit IgG at 0.1 g/mLSubstrate Thermo Scientific Pierce ECL Western Blotting Substrate Thermo Scientific SuperSignal West Pico PLUS substrateDetectionX-ray film with 1 min exposure time X-ray film with 10 min exposure timeWithout enhancer SuperSignal Western Blot EnhancerFigure 54. SuperSignal Western Blot Enhancer reduces background ~1 sec to enable detection of low-abundance targets. K562 cell lysate was loaded into Tris-glycine SDS-PAGE gels at 1.25, 2.5, 5, and 10 g per lane. After electrophoresis, the proteins were transferred to 3 min nitrocellulose membranes.5 minBlocking buffer Thermo Scientific SuperBlock (TBS) Blocking BufferPrimary antibody Mouse anti-ERK1 primary antibody at 1 g/mL15 min Secondary antibody HRP-conjugated goat antimouse IgG at 0.08 g/mLSubstrate Pierce ECL substrateMW 1.25 2.5 5 10 g MW 1.25 2.5 5 10 g DetectionX-ray filmWithout enhancer SuperSignal Western Blot Enhancer-catenin0.25 0.5 1 2 g 0.25 0.5 1 2 g Figure 55. SuperSignal Western Blot Enhancer is compatible with chromogenic and fluorescent detection methods. HeLa cell lysate was separated by electrophoresis, transferred to nitrocellulose (top) or MAPK Thermo Scientific Low-Fluorescence PVDF Membrane (bottom), and western blotting was performed using the conventional method (left) or 0.062 0.125 0.25 0.5 g 0.062 0.125 0.25 0.5 g using the SuperSignal Western Blot Enhancer protocol (right).Top panel Bottom panelBlocking buffer SuperBlock buffer in TBS Blocker BLOTTO in TBSPrimary antibody Rabbit anti-catenin at 0.2 g/mL Rabbit antiMAP kinase at 1 g/mLSecondary antibody AP-conjugated goat antirabbit IgG at 0.04 g/mL Invitrogen DyLight 488conjugated goat antirabbit IgG at 0.1 g/mLSubstrate Thermo Scientific Pierce 1-Step NBT/BCIP Substrate Solution DetectionVisual Typhoon 9410 imagerRecommended productsSuperSignal West Pico PLUS ChemiluminescentInvitrogen goat anti-mouse or goat anti-rabbit Substrate and Pierce ECL Western Blotting SubstrateHRP-conjugated secondary antibodies are are recommended for use with the SuperSignal Westernrecommended for your western blot detection.Blot Enhancer. For chromogenic detection, we recommend Pierce 1-Step NBT/BCIP Substrate Solution. 76'