Application
Western blot
Sample
Human Tissue lysate - whole (Brain)
Loading amount
20 µg
Specification
Brain
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Other product details
Dilution
1/1000
Incubation time
24 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: BSA 5%+tween 0.1%
Secondary antibody
Name
Non-Abcam antibody was used: anti rabbit
Host species: Goat
Clonality: Polyclonal
Conjugation: IRDye® 800CW
Host species: Goat
Clonality: Polyclonal
Conjugation: IRDye® 800CW
Dilution
1/5000
Detection
Detection method
Odyssey
Exposure
6 minute(s) and 0 second(s)
Bands
Specific: 60 kDa Non-specific: 50, 80, 110 kDa
Additional data
Additional Notes
lane1: 1/500
lane2:1/1000
lane3:1/2000
I used BSA 5% as a blocking agent and as a 1 ab diluent for the first three lanes and li-cor buffer for the other 3 lanes.
Insert reads that it detects a band at approximately 65kD. I see nother band around 80kD as well. In our tissue we have seen many times that the molecular weight of the proteins are not exactly as the predicted ones. Since this band runs higher than 60 and the insert said pLIMK antibody detects a band at 65kD this is most likely to be the specific one. Now you say you expect to see a 70-75 kD band, but the insert says it detect a band of approximately 65kD and the image you have in the insert has a band that runs in between 47 and 85, which honestly is really hard to tell wether it is running at 65 or at 75 with that wide of a range.
lane2:1/1000
lane3:1/2000
I used BSA 5% as a blocking agent and as a 1 ab diluent for the first three lanes and li-cor buffer for the other 3 lanes.
Insert reads that it detects a band at approximately 65kD. I see nother band around 80kD as well. In our tissue we have seen many times that the molecular weight of the proteins are not exactly as the predicted ones. Since this band runs higher than 60 and the insert said pLIMK antibody detects a band at 65kD this is most likely to be the specific one. Now you say you expect to see a 70-75 kD band, but the insert says it detect a band of approximately 65kD and the image you have in the insert has a band that runs in between 47 and 85, which honestly is really hard to tell wether it is running at 65 or at 75 with that wide of a range.
Abcam response
Thank you for your review. The band appearing at 60 kDa may be LIM Kinase 1, with phosphorylation at T508. The molecular weights of the bands observed for COL and MCF7 cells, shown on the datasheet, were estimated as 65 kDa but I agree that this is debatable. Another marker between 47 kDa and 85 kDa would improve the estimate.
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 Apr 27 2011