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Cloned sequence is not predicted size by western blot

Cloning Western blot Kilodalton Predicted size

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#1 Rara



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Posted 31 January 2012 - 03:03 PM

Hi, we sub cloned a DNA sequence into our vector of choice. The sequence subcloned was predicted to give a protein molecular weight of 36 kda. When western blot was performed of transduced cells ( expressing the gene of interest), we got a very good western blot signal. However the molecular weight of the protein detected was 48 kda (using a nuMber of different antibodies). Does anyone have an idea of why our cloning is generating a protein that is 10kda heavier than that is predicted by its DNA sequence. It seems unlikely to be phosphorylation etc.. We believe we have completely denatured the protein lunate, including further 'linearisation' using urea.

#2 scolix


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Posted 01 February 2012 - 12:44 AM

posttranslational modifications, not necessarily phosphorylation but could also be glycosylation or any other, seem to be the simplest explanation.

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