The iBlot 3 device delivers consistent results. Note: GAM = goat anti-mouse, HRP = horse radish peroxidase, TBST = tris-buffered saline with Tween™ (solution), GAR = goat anti-rabbit. Local background corrected volume per lysate was plotted for each lane. The iBright FL1500 instrument was used for image capture. SuperSignal West Dura chemiluminescent substrate was used for detection. Primary antibody was removed and GAR-HRP secondary antibody (1:175,000 in TBST) was added to blots and incubated for 2 hours. 4EBP1 primary antibody (1:1,000 in clear milk) was added to blots and incubated for 12 hours at room temperature. Blots were incubated with Pierce Clear Milk Blocking Buffer for 30 minutes. Immunoprocessing was completed using the Bandmate Automated Western Blot Processor. After gel electrophoresis, the proteins were transferred using the method and conditions shown above. Invitrogen iBright FL1500 Imaging System was used for image capture. Thermo Scientific SuperSignal West Dura chemiluminescent substrate was used for detection. Primary antibody was removed and GAM-HRP secondary antibody (1:120,000 in TBST) was added to blots and incubated for 2 hours. Hsp70 primary antibody (1:1,000 in clear milk) was added to blots and incubated for 12 hours at room temperature. Blots were incubated with Thermo Scientific Pierce Clear Milk Blocking Buffer for 30 minutes. Immunoprocessing was completed using the Invitrogen Bandmate Automated Western Blot Processor. The iBlot 3 system demonstrates better transfer efficiency.
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