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Index
00:18
1.
Western Blot - 1_Sample preparation [Video from GeneTex]
00:11
2.
Checking cells
00:12
3.
Remove medium
00:16
4.
Wash cells
00:13
5.
Lyse cells
00:10
6.
Scrape cells
00:09
7.
Collect cells
00:06
8.
Vortex
00:02
9.
Centrifuge
00:12
9.1
Balance the tubes
00:11
10.
Collect supernatant
00:17
11.
Denature
00:09
11.1
Boil the mixture at 95-100 C for 5 min
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00:18
1.
Western Blot - 1_Sample preparation [Video from GeneTex]
00:11
2.
Checking cells
00:12
3.
Remove medium
00:16
4.
Wash cells
00:13
5.
Lyse cells
00:10
6.
Scrape cells
00:09
7.
Collect cells
00:06
8.
Vortex
00:02
9.
Centrifuge
00:12
9.1
Balance the tubes
00:11
10.
Collect supernatant
00:17
11.
Denature
00:09
11.1
Boil the mixture at 95-100 C for 5 min
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Print date : 2023/10/01
賴亮全實驗室
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Western blot (Genetex版本)
Western Blot - 1_Sample preparation [Video from GeneTex]
Duration: 02:33,
Browse: 833,
Last Updated: 2018-12-19
Play Video: http://llai.cm.ntu.edu.tw/media/169
Keypoint
1.
Checking Cells
Before collecting the cells, the cells should be checked microscoptically to ensure they are healthy and growing as expected.
2.
RIPA buffer
RIPA buffer
Tris-HCl (pH 7.6) 25mM
NaCl 150 mM
Triton X-100 1%
Na-deoxychola 1%
SDS 1%
Store at 4C
Add the protease inhibitors immediately prior use
3.
Centrifuge Tip
Use the right size of tubes for the right centrifuge. If tube size is too small, you need to add adaptor.
4.
Centrifuge
20,000 g at 4C for 30 min
5.
Denature
6X SDS Sample Buffer:
Tris-HCl (pH6.8) 315.5mM
Glycerol 50%
SDS 0.1 g/ml
bromophenol blue 0.05%
2-b mercaptoethanol 10%
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Details
00:18
1.
Western Blot - 1_Sample preparation [Video from GeneTex]
00:11
2.
Checking cells
00:12
3.
Remove medium
00:16
4.
Wash cells
00:13
5.
Lyse cells
00:10
6.
Scrape cells
00:09
7.
Collect cells
00:06
8.
Vortex
00:02
9.
Centrifuge
00:12
9.1
Balance the tubes
00:11
10.
Collect supernatant
00:17
11.
Denature
00:09
11.1
Boil the mixture at 95-100 C for 5 min
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Q1.
When to add protease inhibitors?
Q2.
What is the setting of centrifuge?
Q3.
Collect the supernatant and place in a fresh tube kept on ___?
Q4.
What is the purpose of adding sample buffer?
Location
Media Center
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Western blot (Genetex版本)
Folder name
Western blot (Genetex版本)
Author
賴亮全
Branch
賴亮全教授
Created
2018-12-19 18:18:30
Last Updated
2018-12-19 22:33:54
Browse
833
Duration
02:33
Reference
1
Media Center
...
Western blot (Genetex版本)
1.
Western Blot - 1_Sample preparation [Video from GeneTex]
2.
Western Blot - 2_Electrophoresis [Video from GeneTex]
3.
Western Blot - 3_Semi-Dry transfer [Video from GeneTex]