Performance of a Western Blot

A step-by-step overview providing detailed insight into the resolution of complex protein mixtures by SDS-PAGE prior to the detection of proteins of interest by highly sensitive chemiluminescent western blot. 0:00 Introduction 0:05 SDS-PAGE gels come with different well formats 0:19 Assembly of the running apparatus 0:33 Addition of running buffer 0:54 Sample loading into individual wells 1:43 Apparatus assembly followed by running of the gel at 150V 1:55 Apparatus disassembly following completion of the run 2:10 Removing individual gels 2:25 Removal of the precast acrylamide gel 2:51 Removal of the stacking gel and dye front 3:12 Equilibration of the gel in transfer buffer 4:12 Addition of the upper pad 4:22 Removing trapped air 4:32 Transfer cassette assembly prior to transfer at 20V for 7min. 4:40 Cassette disassembly post transfer 5:02 Blocking of the membrane in blocking for 1hr at ambient temperature 5:09 Membrane preparation and addition primary antibody (1:50 - 1:2000) 6:47 ambient temperature on an orbital shaker 7:31 1:40000 and incubate for 1hr at am temperature on an orbital shaker 8:36 Washing of the membrane prior to the addition of substrate 9:13 Focussing and imaging the membrane

• #science_technology
• #wb
• #western_blot
• #sds_page
• #sds
• #antibody
• #antibodies