Application
Western blot
Sample
Mouse Cell lysate - other (MEF)
Loading amount
50 µg
Specification
MEF
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 3%
Other product details
Dilution
1/1000
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: TBST
Secondary antibody
Name
Non-Abcam antibody was used: goat anti rabbit
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/2000
Detection
Detection method
ECL
Exposure
3 second(s)
Bands
Specific: 23 kDa
Positive control
I re-confirmed the experiment with lysates from Jurkats (positive control), MEFs and HELAs (negative control), and all samples gave a single strong band at 23 KD. Between this result, your mentioning that immunogen used to raise the antibody is not conserved in mice, and the fact that BIK should appear between 18-20Kd, I suspect if the antibody is recognizing BIK to begin with.
Additional data
Additional Notes
The mouse cells were not transfected with the human protein. Attached is a modified image where the positive control (Jurkat cells -Lane 1) was run next to cells of mouse origin (Lane 2)
The contents from the first lane did not spill into the next lane. The sample volume loaded onto the wells was just 20uL and there was no chance of accidental spilling.
The contents from the first lane did not spill into the next lane. The sample volume loaded onto the wells was just 20uL and there was no chance of accidental spilling.
Abcam response
The Western blot looks very nice, however the alignement between the mouse and the human protein is very low in the immunogen region (around Threonine 33). We have therefore not added and do not guarantee crossreactivity of this antibody with the mouse protein.
We would be very interested in feedback if anyone else has used this antibody on mouse as well. Thank you very much.
We would be very interested in feedback if anyone else has used this antibody on mouse as well. Thank you very much.
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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提交于 Feb 24 2011