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stable transfectants


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#1 Irene

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Posted 20 March 2009 - 08:23 AM

hi, I have been able to obtain a stable transfectant of my mutant gene in HEK293 cells. the thing is that when i have checked the expression pattern by WB the molecular weight has slightly changed and has appeared a double band. Could it be because of an abnormal integration in the genome? or because a postransductional modification??

thanks

#2 Dr Teeth

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Posted 20 March 2009 - 08:33 AM

hi, I have been able to obtain a stable transfectant of my mutant gene in HEK293 cells. the thing is that when i have checked the expression pattern by WB the molecular weight has slightly changed and has appeared a double band. Could it be because of an abnormal integration in the genome? or because a postransductional modification??

thanks


Could be post-translational. Is the lower band the expected MW? How different are the two bands in size?

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 Irene

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Posted 20 March 2009 - 08:51 AM

hi, I have been able to obtain a stable transfectant of my mutant gene in HEK293 cells. the thing is that when i have checked the expression pattern by WB the molecular weight has slightly changed and has appeared a double band. Could it be because of an abnormal integration in the genome? or because a postransductional modification??

thanks


Could be post-translational. Is the lower band the expected MW? How different are the two bands in size?


The lower ban is more or less the expected one but not exactly and the size is similar for both

#4 Dr Teeth

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Posted 20 March 2009 - 09:41 AM

I mean, specifically, how similar are they? 1 kD, 5 kD? etc. Do you have a positive control? When you transiently transfect, which band do you see? If you haven't done so already, re-run the gel comparing the lysates from your stable, transiently transfected, and negative control samples and compare the MW of your target protein. Is the second band higher and only appearing in your stable line? If so, it might be post-translational modification. Are there any known or predicted modifications such as sumoylation, etc. for your gene?

Edited by Dr Teeth, 20 March 2009 - 09:44 AM.


Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#5 Irene

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Posted 20 March 2009 - 09:59 AM

I mean, specifically, how similar are they? 1 kD, 5 kD? etc. Do you have a positive control? When you transiently transfect, which band do you see? If you haven't done so already, re-run the gel comparing the lysates from your stable, transiently transfected, and negative control samples and compare the MW of your target protein. Is the second band higher and only appearing in your stable line? If so, it might be post-translational modification. Are there any known or predicted modifications such as sumoylation, etc. for your gene?



The bands are similar and they are separated around 2-3 KDa. I have run both positive and negative controls and the bands are nearly the positive control of my band but not exactly the same weight. I have also checked that transiently transfectants does not show the double band, and nor do does some other stable transfectants i have obtained in other cell system (c6 cells). My protein is suppossed to be phosphorylated, and it is also very hydrofobic (we stronlgy believe it can form dimers). I have tried to dephosphorylate my protein using Alkaline phosphatase to see if the double band dissapeared but it doesn“t, but the protein was phosphorylated because it changed the mobility.

thanks for your help”””

#6 Dr Teeth

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Posted 20 March 2009 - 10:42 AM

Phosphorylation is not likely to change your MW enough to see on a Western and dimers would not be occuring under the denaturing conditions of an SDS-PAGE.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
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#7 Irene

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Posted 20 March 2009 - 11:03 AM

Phosphorylation is not likely to change your MW enough to see on a Western and dimers would not be occuring under the denaturing conditions of an SDS-PAGE.



Yes, I know that, the pattern of dephodphorylation is not very dramatically changed, and as we can see the dimer even in SDS-PAGE is why we thought the protein so very hydrofobic.




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