There are two sepcific and strong bands when I use anti-myc(santa cruz 9E10) to analysis the whole cell lysates of transfected flag-myc 293t cell.But I could not detect any specific bands when I analysis the whole cell lyasates of HELA, HCT116,U87,MEF cell.The process of lysing is as below
1,wash the plates twice with cold PBS
2.add 200μl RIPA buffer into plates and scratched the cell into a tube
3.incubate the tube on ice for 20min
4.sonic 5s and break 2s for 12 times
5.centrifuge 13500 rpm for 15min,4℃
6. using supernants for 10% SDS-page immunoblot
Are there any problems?
does anybody konw how to successfuly detect endogenous myc protein?
Should I pay attention to some details specially?
The 9E10 antibody is good for detecting the myc-tag usually present in expression vectors. We had problems earlier in detecting mouse c-myc using this antibody. Finally the conclusion was that these antibodies could detect myc-tag and human-c-myc (to some extent / I have not tried) but cannot detect mouse c-myc. We had then used c-Myc (N-262) from Santacruz to detect endogenous c-myc in MEFs.
Hope this helps :Manoj
It is likely that your experimental conditions to detect Myc protein that is over-expressed in transfected HEK293 cells are appropriate. However, to detect the endogenous much lower levels of c-Myc one needs to optimize the experimental conditions. This would include total cell extracts in your experimental conditions from actively proliferating cultures (the Myc is inducible in an early G1 phase of the cell cycle) and immunoblotting conditions (PVDF membrane and reduced dilution of the anti-Myc antibody).
The 9E10 antibody is good for detecting the myc-tag usually present in expression vectors. We had problems earlier in detecting mouse c-myc using this antibody. Finally the conclusion was that these antibodies could detect myc-tag and human-c-myc (to some extent / I have not tried) but cannot detect mouse c-myc. We had then used c-Myc (N-262) from Santacruz to detect endogenous c-myc in MEFs.
Hope this helps :Manoj
You need to protect the c-myc from deteriorating in your procedures AT ALL TIMES. use temperatures a little lower than you currently use (try that first), and if not, then use a very weak neutral detergent in all your solutions AT ALL TIMES. A weak neutral detergent will not denature the proteins but instead protect them from denaturing conditions as already exist in your procedures. Good luck, these two procedures WILL NOT negatively affect any of the results of your procedures.
Endogenous myc-levels may be to low to detect in whole cell lysate. Try to immunoprecipitate myc before analysing by western blot. I had the same problem with an other protein, I could detect the overexpressed and immunoprecipitated protein but not the endogenous protein. Good luck.
We have been working with Myc in more than 20 years. Myc could be difficult to detect in many cell lines due to the level of the endogenic protein. In case you want to immunoprecipitate Myc then running WB you could Ip with N-262 and for WB use C-33 but biotinylated to reduce the detection of Ig bands. and as secondary add Streptoavidin. Check many of our publications.
Good Luck
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