Transfer proteins to nitrocellulose membrane
.
-
00:00
1.
Western Blot - 3_Semi-Dry transfer [Video from GeneTex]
-
00:18
2.
Transfer proteins to nitrocellulose membrane
-
00:31
2.1
equilibrate in transfer buffer for 15 min
-
00:39
2.2
Rinse 4 pieces of blot paper with transfer buffer
-
00:53
2.3
Place 2 pieces of pre-soaked blot paper onto the platinum anode
-
01:02
2.4
Roll a test tube over the layer to remove bubbles
-
01:05
2.5
Place the pre-wetted nitrocellulose membrane on top of the wetted blot paper
-
01:14
2.6
Place the equilibrated gel on top of the nitrocellulose membrane
-
01:23
2.7
Place 2 pieces of pre-wetted blot paper on top of the gel
-
01:28
2.8
Roll a test tube over the layer to remove bubbles
-
01:31
2.9
Sop up transfer buffer with paper towel
-
01:42
2.10
Place the cathode plate onto the stack
-
01:51
2.11
Place safely cover on the unit
-
01:56
2.12
Run the transfer unit at 18V for 1 h
-
02:13
2.13
Rinse the membrane with H2O
-
02:19
3.
Check for success of transfer
-
02:19
3.1
Stain the nitrocellulose with Ponceau Red solution
-
02:36
3.2
Wash in H2O unter the water is clear and protein bands are well-defined
-
02:47
3.3
The membrane can be destained by washing in TBST or water
-
02:58
4.
Block the membrane
-
02:58
4.1
Incubate the membrane in blocking buffer at RT for 30-60 min
-
03:05
4.2
Pour off the blocking buffer
-
03:07
5.
Incubate with the primary antibody
-
03:07
5.1
Incubate the membrane in blocking buffer with primary antibody at 4C overnight
-
03:17
6.
Wash the nitrocellulose membrane
-
03:17
6.1
Pour off the primary antibody solution
-
03:20
6.2
Wash the membrane 5 times for 3-5 min each time with TBST
-
03:34
7.
Incubate with the secondary antibody
-
03:34
7.1
Incubate the membrane in blocking buffer with 2nd antibody at RT for 1 h
-
03:49
8.
Wash the nitrocellulose membrane
-
03:49
8.1
Pour off the 2nd antibody solution
-
03:52
8.2
Wash the membrane 5 times for 3-5 min each time with TBST
-
04:07
9.
Detect the proteins using detection system
-
04:07
9.1
Remove excess TBST
-
04:14
9.2
Follow the instruction of ECL kit
-
04:18
9.3
Mix equal vol. of Enhanced Luminol Reagent (A) and the Oxidizing Reagent (B)
-
04:34
9.4
Pour the chemiluminescence reagent into the membrane (incubate 1 min at RT)
-
04:43
9.5
Take a picture with CCD camera
- Index
- Notes
- Comment
- Fullscreen
Western Blot - 3_Semi-Dry transfer [Video from GeneTex]
Duration: 05:05, Browse: 1153, Last Updated: 2018-12-20
Play Video: http://llai.cm.ntu.edu.tw/media/171
Keypoint
- 1.
- 2.Transfer proteins to nitrocellulose membrane.
- 3.Check for success of transfer.
- 4.Check for success of transfer.
- 5.Block the membrane.
- 6.Wash the nitrocellulose membrane.
- 7.Incubate with the secondary antibody.
-
00:00
1.
Western Blot - 3_Semi-Dry transfer [Video from GeneTex]
-
00:18
2.
Transfer proteins to nitrocellulose membrane
-
00:31
2.1
equilibrate in transfer buffer for 15 min
-
00:39
2.2
Rinse 4 pieces of blot paper with transfer buffer
-
00:53
2.3
Place 2 pieces of pre-soaked blot paper onto the platinum anode
-
01:02
2.4
Roll a test tube over the layer to remove bubbles
-
01:05
2.5
Place the pre-wetted nitrocellulose membrane on top of the wetted blot paper
-
01:14
2.6
Place the equilibrated gel on top of the nitrocellulose membrane
-
01:23
2.7
Place 2 pieces of pre-wetted blot paper on top of the gel
-
01:28
2.8
Roll a test tube over the layer to remove bubbles
-
01:31
2.9
Sop up transfer buffer with paper towel
-
01:42
2.10
Place the cathode plate onto the stack
-
01:51
2.11
Place safely cover on the unit
-
01:56
2.12
Run the transfer unit at 18V for 1 h
-
02:13
2.13
Rinse the membrane with H2O
-
02:19
3.
Check for success of transfer
-
02:19
3.1
Stain the nitrocellulose with Ponceau Red solution
-
02:36
3.2
Wash in H2O unter the water is clear and protein bands are well-defined
-
02:47
3.3
The membrane can be destained by washing in TBST or water
-
02:58
4.
Block the membrane
-
02:58
4.1
Incubate the membrane in blocking buffer at RT for 30-60 min
-
03:05
4.2
Pour off the blocking buffer
-
03:07
5.
Incubate with the primary antibody
-
03:07
5.1
Incubate the membrane in blocking buffer with primary antibody at 4C overnight
-
03:17
6.
Wash the nitrocellulose membrane
-
03:17
6.1
Pour off the primary antibody solution
-
03:20
6.2
Wash the membrane 5 times for 3-5 min each time with TBST
-
03:34
7.
Incubate with the secondary antibody
-
03:34
7.1
Incubate the membrane in blocking buffer with 2nd antibody at RT for 1 h
-
03:49
8.
Wash the nitrocellulose membrane
-
03:49
8.1
Pour off the 2nd antibody solution
-
03:52
8.2
Wash the membrane 5 times for 3-5 min each time with TBST
-
04:07
9.
Detect the proteins using detection system
-
04:07
9.1
Remove excess TBST
-
04:14
9.2
Follow the instruction of ECL kit
-
04:18
9.3
Mix equal vol. of Enhanced Luminol Reagent (A) and the Oxidizing Reagent (B)
-
04:34
9.4
Pour the chemiluminescence reagent into the membrane (incubate 1 min at RT)
-
04:43
9.5
Take a picture with CCD camera
- Location
-
- Folder name
- Western blot (Genetex版本)
- Author
- 賴亮全
- Branch
- 賴亮全教授
- Created
- 2018-12-19 18:19:29
- Last Updated
- 2018-12-20 08:02:15
- Duration
- 05:05
- Reference
- 1