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Protein much larger on Western blot than predicted


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12 replies to this topic

#1 Lusen

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Posted 04 March 2009 - 05:49 AM

Anybody who has got an idea why my protein seems to be running at around 90 kDa when it is predicted (based on the sequence) to be around 50 kDa. I am running on a normal SDS-PAGE gel (reducing and denaturing). I am pretty sure that the antibody works since knock outs lacks the band and all controls have it.

p.s. It is a nuclear receptor (RXR) it that helps with the answer ???

#2 madrius1

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Posted 04 March 2009 - 07:39 AM

A (very) quick search in Pubmed retrieved a posttranslational modification (sumoylation) of RXR. There could be other too, so that may explain the shift of the band.

#3 Lusen

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Posted 04 March 2009 - 08:14 AM

A (very) quick search in Pubmed retrieved a posttranslational modification (sumoylation) of RXR. There could be other too, so that may explain the shift of the band.


Yes I looked into that, but sumoylation as far as I have read often affects only a smaller fraction of a given protein pool and I dont see any in the right size. I think there was another reason for discarding this modification, but I cant remember right now.

#4 HomeBrew

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Posted 04 March 2009 - 08:46 AM

If you're using a pre-stained (colored) ladder to estimate MW, that could throw your results off -- these ladders are not useful for size estimation, because the migration the various bands in the ladder exhibit (the relative MW) is altered by many factors, including the pH, gel concentration, gel system and running conditions.

#5 Lusen

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Posted 05 March 2009 - 01:28 AM

If you're using a pre-stained (colored) ladder to estimate MW, that could throw your results off -- these ladders are not useful for size estimation, because the migration the various bands in the ladder exhibit (the relative MW) is altered by many factors, including the pH, gel concentration, gel system and running conditions.


I use one prestained and one unstained....same result

#6 aimikins

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Posted 05 March 2009 - 08:30 AM

are you certain you're not getting dimers?
"it is a miracle that curiosity survives formal education" -A.E.

#7 HomeBrew

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Posted 05 March 2009 - 02:20 PM

Wrong start codon? Or, if you're working in eucaryotes, a splice variant?

#8 Lusen

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Posted 06 March 2009 - 04:34 AM

are you certain you're not getting dimers?


Can I get that on a denaturing and reducing gel ?

#9 Lusen

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Posted 06 March 2009 - 04:38 AM

Wrong start codon? Or, if you're working in eucaryotes, a splice variant?


Possibly, but these receptors are very conserved and the predicted size fits all the alignments and sizes of related and very distantly related organisms.
Thanks to everybody for suggestions, guess I might come back later with an answer if I find the solution

#10 Dr Teeth

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Posted 06 March 2009 - 08:40 AM

Post-translational modifications should not alter your target molecular weight by 40 kD and dimers would not be occuring under denaturing conditions. A splice variant would be more likely, though still such a big size difference would not be expected for receptors like RXR.
Do you have a positive control? Try to include a sample with in vitro translated RXR protein or another protein known to be ~ 50 kD.

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

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Posted 10 March 2009 - 10:43 AM

This all very strange, also reading the other thread " MW marker shows wrong MWs?"

I havent really had this type of problems before. I use invitrogen gels and buffers and buy markers that should fit with the types of gels I use. Stained and unstained.

I have run other samples on the same gels as the RXR samples and they seem to be running at predicted size. How would I get hold of an in vitro translated RXR ?

Generally this all boils down to whether my antibody is really recognizing the right protein. The only real reason for me believing this (apart from that this is what it was designed for) is the lack of bands in KO animals.

#12 mikew

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Posted 10 March 2009 - 12:08 PM

So, this problem keeps creeping up on this site.
The question is have you done your Western with a negative control?
Use a cell line that has no RXR to see if what you are seeing is real.
The truth is that you have no idea whatsoever if the bad you are detecting is RXR
unless you do controls. Positive, negative, etc.
Transfect the cells with RXR, look for overexpression. Knock down RXR lown for its disappearance.
You cannot, absolutely cannot assume the bands you are seeing on a blot are the band you think it is.
Especially if this antibody is from Santa Cruze. These antibodies are often of low quality.
I have Western blotted for RXR previously and have never achieved a good result. Maybe the newer antibodies are better.
Many antibodies detect non-speific bands. If you Western is showing a band of aberrant size, most likely your antibody is bad, the gel was made improperly or your Western technique doesn't work. It reeally isn't anything complex.

#13 hann.a

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Posted 30 April 2009 - 05:08 AM

Post-translational modifications should not alter your target molecular weight by 40 kD and dimers would not be occuring under denaturing conditions. A splice variant would be more likely, though still such a big size difference would not be expected for receptors like RXR.
Do you have a positive control? Try to include a sample with in vitro translated RXR protein or another protein known to be ~ 50 kD.


I'm working with membrane proteins too - and we regular see dimers, even under denaturing conditions. So the 90KDa could very well be a dimer.
Also, check out the following paper (they also have examples of proteins running as mutlimers on denaturing SDS-PAGE - with very out of range MWs):
"Detergent binding explains anomalous SDS-PAGE migration of membrane proteins"
Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM. Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5.




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