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Trouble estimating my protein size using ladder (image included)


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2 replies to this topic

#1 Fluffy

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Posted 10 December 2012 - 07:25 PM

Hello Everybody,

I transformed my pET14b vector containing my target insert into Bl21(DE3) cells and induced my culture with IPTG. I ran an SDS-PAGE to check for the solubility, expression, and whether my protein got induced. The different fractions cellular fractions I ran on gel include total cell protein fraction and soluble cytoplasmic fraction.

My problem is I cannot locate my protein on the SDS-PAGE gel. My protein size is approximately 21.38 kilodaltons. I cannot really know how my inductions really went? Can anyone provide me with feedback please. I would greatly appreciate it.

My image organization
far left-ladder, total cell protein induced, total cell protein uninduced, soluble cytoplasmic induced, soluble cytoplasmic uninduced-far right.
Here is the image12.10.2012 SDS UBC1.jpg

For ladder image here is the link http://tools.invitro...ned_Std_man.pdf

Thanks again!

#2 John Forsberg

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Posted 10 December 2012 - 08:03 PM

As I've never used those markers before, I'm not sure I'm confident of the band I'm pointing out, but if your markers are what I think they are I'd say your protein is the band in the induced total and soluble lanes that is just above the third obvious marker from the bottom.

I've attached the band I'm thinking is correct, and what I think the markers are. I can't be positive, as I usually use multicolored markers, and I usually get a feel for how the markers run on a given gel type over time, so I can tell at a glance which markers are which.

I'll also note that with many protein expressions that I do, I do not see my protein in the total extract at all, but only see it after enrichment after binding to affinity resin and eluting (for many His-tagged proteins in particular). So you may want to try a trial run of your first purification step to see if you get a more prominent band at the right size.

12.10.2012 SDS UBC1 (1).jpg

#3 Fluffy

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Posted 12 December 2012 - 11:28 AM

Thanks for your reply John.

Ok I am waiting on my anti-his antibody and purification kit and will do as suggested. I hope for the best.
Thanks again :)




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