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Western Blot: protein band did not appeared at the correct location

mouse iNOS protein

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#1 shiwei

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Posted 11 September 2014 - 10:57 PM

Hi all, need some help/ advises for a problem I encounter as mentioned in the title.

 

I am detecting iNOS (inducible nitric oxide synthase) protein from a murine cell line. After the western blot, I can see a clear and reproducible band. However, the band did not appeared at the expected location. the band is approximately 80kDA smaller that expected (expected molecular weight of the iNOS protein is 130kDA). 

 

Does anyone know what may be the cause of the above phenomena? Is it possible that the protein ladder which I use is misleading?

Please kindly advise.

 

Thank you so much.smile.png 

 

*attached are the picture of the blot membrane and the pre-stained protein ladder.

  -lane 1 and 3: unstimulated cells sample

  -lane 2 and 4: stimulated cells sample

  -theoretically, stimulated cells will have higher iNOS expression than unstimulated one. the bands appreared showed the same trend.         However, the bands appeared at different location than the expected location. 

Attached Thumbnails

  • iNOS-forum 2.jpg
  • ladder-forum2.jpg


#2 mlomonaco

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Posted 12 September 2014 - 08:15 AM

Could this be IgG?



#3 bob1

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Posted 13 September 2014 - 01:38 PM

Check that the antibody is suitable for use in mouse, check that it is suitable for western blots. What % gel are you using?

 

If you haven't already, titrate the antibody to ensure that you are using the correct concentration. Try also loading more and less protein to see if that makes any difference. 

 

Note also that proteins often don't run at the predicted size, but it would be unusual to get such a big difference.  Check the literature to see if this is a known problem for iNOS.

 

Unless you have immunoprecipitated the protein or are using a cell that produces antibodies, IgG is unlikely to show on the blot and would usually be around 50 kDa for heavy chain.






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