I have a problem using the Drp1 C5 and p-Drp1 S616 antibodies from Santa Cruz for the western blotting technique. The band should be at around 70-80kD, so I used 10% gel. I used to transfer 90 V 110 min in the PDVF membrane. I used 35 ug protein. The primary antibody i used to 48 h incubation 4 degrees and consequently 1 h for the secondary antibody. but my band looks like a smear. Not clear though I used high exposure.
Hello Nazmul Islam
Usually the primary antibody incubation period is overnight (16 hours) at
4 degree C. Why have you kept it for 48 hours incubation period ? Any specific reason.
Hello Malcolm Nobre
Actually previously I also used to 16 h incubation but later I tested several hour in different antibody. I got some antibody come good result in more than 20 h . So i used this drp1 also in 48 h . But still i need to find out the mechanism.
Hi Nazmul Islam . I would look into getting a primary from a different source. SC is not known for quality Abs.
Hi, The incubation showed the the pattern of aspecific binding of primary antibody. I agree with the subbestion above to try using other company. If you can't buy another one in this case read on the datasheet the cells used by santacruz to perform the western (in general they showed the WB that they made) and if find it to have at least a good positive control. I used a positive control when I was in your same situation, anyway if the primary antibody it is not good and dn't bind your protein in you cells you don't have a lot of possibililities.
I don't known this protein but in your system is it well expressed from previous work? Is it cytosolic or membrane protein? You can improve the extraction method?
Valeria
In my opinion, the antibody is not specific (for ref. see Baker M. Reproducibility crisis Blame it on the antibodies. Nature. 2015_521.p274-6. doi 10.1038521274a). And/or other methodological uncertainties cound be the reason (e.g. apparature, experimentator etc.).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line