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Phone call enquiring whether the Western blot on the dateasheet for ab70134 was produced using ectopic expression as the datasheet states the expected size is 77 kDa whilst a report by Jiang et al, EMBO 1999 states that endogenous expression protein size of 100-150 kDa is observed. |
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ANSWER: |
Thank you for contacting us in regards to Anti-DBF4 antibody (ab70134). I have been assured by the source of this antibody that the Western blot performed and displayed on the datasheet was performed with endogenous expression of DBF4 from NIH3T3 and COS7 cells treated with H2O2 and PMA respectively. As you have pointed out this conflicts with the report by Jiang et al, EMBO 1999. I am currently looking into other reports of DBF4 expression to see if this difference may be as a result of postranslational modification differences between the two experiments. As soon as I have any further information I will let you know. |
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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
All lanes : Anti-DBF4 antibody (ab70134) at 1/500 dilution
Lane 1 : NIH3T3 cell extracts
treated with H2O2 (100µM, 30mins) at 5 µg
Lane 2 : COS7 cell extracts
treated with PMA (125mg/ml, 30mins)
Lane 3 : NIH3T3 cell extracts
treated with H2O2 (100µM, 30mins) and PMA (125mg/ml, 30mins) with immunising peptide at 5 µg
Predicted band size : 77 kDa
Observed band size : 77 kDa
Additional bands at : 60 kDa. We are unsure as to the identity of these extra bands.
ICC/IF image of ab70134 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab70134, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
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