Application
Western blot
Sample
Mouse Cell lysate - whole cell (BMDCs, Whole spleen lysate and CY15 (DC cell line))
Loading amount
75 µg
Specification
BMDCs, Whole spleen lysate and CY15 (DC cell line)
Treatment
Treated or not to thapsigargin
Gel Running Conditions
Reduced Denaturing (10 %)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Other product details
Dilution
1/2000
Incubation time
18 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: Milk 2,5 %, found optimal over BSA
Secondary antibody
Name
Non-Abcam antibody was used: Dako anti-Rabbit, HRP-linked
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/10000
Detection
Detection method
ECL+
Exposure
20 minute(s) and 0 second(s)
Bands
Specific: ~39 kDa Non-specific: 4 bands 25-37 kDa, 4 kDa
Positive control
Thapsigargin-treated cells O/N
Negative control
Non-treated cells
Additional data
Additional Notes
The band of 39 kDa is recognized but background signal is too strong to properly isolate the signal of the specific signal.
On the image, the results show the cell lysate from CY15 cells, a cell line of immature DCs, but simimar results were obtained with spleen lysate.
Legend of the image:
Gels
1: Block Milk 5%: 1st Ab 1/1000 in Milk 2,5%: 2nd Ab 1/10000 in 2,5 % Milk
2: Block Milk 5%: 1st Ab 1/2000 in BSA 1,5%: 2nd Ab 1/20000 in 2,5 % Milk
3: Block Milk 5%: 1st Ab 1/2000 in Milk 2,5%: 2nd Ab 1/10000 in 2,5 % Milk
4: Block Milk 5%: 1st Ab 1/1000 in Milk 2,5%: 2nd Ab 1/20000 in 2,5 % Milk
5: Block Milk 5%: 1st Ab 1/2000 in BSA 1,5%: 2nd Ab 1/50000 in 2,5 % Milk
6: Block Milk 5%: 1st Ab 1/2000 in Milk 2,5%: 2nd Ab 1/20000 in 2,5 % Milk
7: Block Milk 5%: 1st Ab 1/4000 in BSA 1,5%: 2nd Ab 1/20000 in 2,5 % Milk
8: Block Milk 5%: 1st Ab 1/4000 in BSA 1,5%: 2nd Ab 1/50000 in 2,5 % Milk
All gels of this experiment loaded with 75µg proteins.
On the image, the results show the cell lysate from CY15 cells, a cell line of immature DCs, but simimar results were obtained with spleen lysate.
Legend of the image:
Gels
1: Block Milk 5%: 1st Ab 1/1000 in Milk 2,5%: 2nd Ab 1/10000 in 2,5 % Milk
2: Block Milk 5%: 1st Ab 1/2000 in BSA 1,5%: 2nd Ab 1/20000 in 2,5 % Milk
3: Block Milk 5%: 1st Ab 1/2000 in Milk 2,5%: 2nd Ab 1/10000 in 2,5 % Milk
4: Block Milk 5%: 1st Ab 1/1000 in Milk 2,5%: 2nd Ab 1/20000 in 2,5 % Milk
5: Block Milk 5%: 1st Ab 1/2000 in BSA 1,5%: 2nd Ab 1/50000 in 2,5 % Milk
6: Block Milk 5%: 1st Ab 1/2000 in Milk 2,5%: 2nd Ab 1/20000 in 2,5 % Milk
7: Block Milk 5%: 1st Ab 1/4000 in BSA 1,5%: 2nd Ab 1/20000 in 2,5 % Milk
8: Block Milk 5%: 1st Ab 1/4000 in BSA 1,5%: 2nd Ab 1/50000 in 2,5 % Milk
All gels of this experiment loaded with 75µg proteins.
Abcam response
We appreciate customer's feedback.
We have not tested Mouse species. However, it could have been a vial problem. Hela cells would be a good positive control to be sure that the antibody is working.
We have not tested Mouse species. However, it could have been a vial problem. Hela cells would be a good positive control to be sure that the antibody is working.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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投稿 Jun 16 2010