Performance of a Western Blot
43.3 هزار بار بازدید -
12 سال پیش
-
A step-by-step overview providing detailed
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
12 سال پیش
در تاریخ 1391/09/06 منتشر شده
است.
43,342
بـار بازدید شده