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Problem with removing fusion protein. MALDI TOF. Tricine SDS PAGE.


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#1 L u c a

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Posted 26 March 2012 - 08:05 AM

Hello,
I have problem with cleave a peptide (3,2 kDa) off a fusion protein.The construct contains protease site between fusion protein and the peptide of interest (actually I have 2 constructs: with enterokinase and with factor Xa site). Both proteases seem to work, but both show some nonspecificity. I use tricine sds gel, that is for visualising small peptides, but I don’t get a band at the needed position. The same result is with MALDI TOF. Maybe the concentratoin of product is too low to visualise it on gel, but I geally worry about MALDI result because it shows nothing at 3,2 kDa.. Honestly it barely shows something... I guess any contaminant may interrupt. Probes contain 1 M urea, 20-50 mM Tris, 1- 2 mM CaCl2, 0,1% Tween20, 100 mM NaCl and some imidasole.. Can any of this reagents interrupt MALDI? Or may you share some experience about tricine gel preparation which could help me?

P.S. There is no problem with MALDI apparatus and matrix. They do their work well.

#2 neuron

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Posted 30 March 2012 - 02:18 AM

Hi,

I am trying to understand your experiment, please correct me if I am wrong. you have a fusion protein, you have cleaved the 3.2kda peptide from this protein and now you have loaded it onto tris tricene gel to visualize it. But you don't see any band at 3.2 Kda , still you cut the gel, extract the peptide and spot it on MALDI plate. Am I correct?

In my personal experience, identifying a small protein on MALDI is difficult. What I would suggest you is to clean up your sample with zip-tip or so and then give it a try.

Hope it helps Posted Image

amj2

Edited by amj2, 30 March 2012 - 02:19 AM.





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