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why i get double band for recombinant protein


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9 replies to this topic

#1 thanhtam

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Posted 05 August 2013 - 01:56 AM

Hi all,

I am trying to express a protein fuse with His-tag in E.coli. When purify it by Ni-NTA gel or run western blot, i always get two bands which only have a few kDa different. Why i get band like that and how I can make sure which band is my protein?



#2 loz

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Posted 11 August 2013 - 03:22 AM

silly question....but it isn't a dimer or something like that is it?



#3 loz

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Posted 11 August 2013 - 03:30 AM

Actually, I remember purifying a protein once that was not a dime and it always showed a doublet via western blot. We could not explain it really, but it was put down to gycosylation for some reason which did not ever make sense to me!



#4 loz

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Posted 11 August 2013 - 03:32 AM

can you use akta or mass spec?



#5 Adrian K

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Posted 11 August 2013 - 08:38 AM

Suggestion: Possible self-cleavage protein

 

Try analyze your protein using SignalP at http://www.cbs.dtu.d...rvices/SignalP/ or TargetP http://www.cbs.dtu.d...rvices/TargetP/ or peptide cutter at http://web.expasy.org/peptide_cutter/

 

Using gel filtration chromatography system, we usually can see If is the case of dimer or multimers. However for SDS-PAGE and WB, since we had boiled and denatured the proteins, all the bondings that hold the multimers should be destroyed, and I believe even if there are presence of multimers, through SDS-PAGE or WB, you only can see 1 single band in the end results.

 

Hope this helps.


Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

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#6 loz

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Posted 12 August 2013 - 02:56 AM

That is very interesting Adrian.

I work with an excreted trans membrane protein that is a dimer, the one where I mentioned this doublet (I call it a doublet because it is not the correct size to be the dimeric partner, definitely something else as it is present with bothe monomer and dimer bands). In one vector in particular, it always produces a doublet via W.B, whether this is a reducing or a non reducing gel. I know that this is not my question, but who knows, might help solve the quandary. Interesting hey?



#7 Adrian K

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Posted 12 August 2013 - 09:28 AM

Correct me if I am wrong, My impression is you are using Anti-His for your WB detection? Or do you mean you are using specific some antibody?

Just curious, can you detail to us your primary and secondary antibody origins?

 

Based on what you mentioned, I am thinking the possibility of your recombinant protein might be forming a complex with something else. Possible for you to isolate the two proteins and perform mass spec analysis?


Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#8 littlebull

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Posted 19 August 2013 - 08:46 AM

Which system have you used for protein expression? There may be modification, such as glycosylation.  



#9 guyava

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Posted 21 August 2013 - 12:39 AM

assuming your expression system is eukaryotic, this is probably a PTM, most likely glycosylation. To get a definitive answer you can:

1. Excise the 2 bands from the gel an send to MS. This will tell you if these 2 bands are actually the same protein or not.

2. Incubate you protein with a deglycosylation enzyme (can be purchased from Sigma or elsewhere) and test the disappearance of one of the bands (most likely the upper one) - which will indicate that the doublet is due to heterogenous glycosylation.



#10 labtastic

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Posted 29 August 2013 - 05:23 PM

Had the exact same problem in graduate school. Super super long story short...turns out the lower band was the protein with an internal crosslink. Upper band was non-crosslinked. 






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