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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 - 08:07 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 proteaMatt

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Posted 28 March 2012 - 06:51 AM

Urea, Tris, and Tween are all going to cause major problems for MALDI analysis, and CaCl2 and NaCl (especially at that concentration) can also suppress ionization to an extent. The Urea and Tris are likely going to need to be removed by a clean-step such as acetone or acetonitrile precipitation, dialysis, ultrafiltration, or some type of RP clean up (either by an LC system or by functionalized pipete tips), most of these methods would help to remove CaCl2 and NaCl as well. Surfactants in general are a big problem for MALDI and there are various work arounds that exist. One thing you could try, would be to replace the Tween 20 in your solution with an acid labile surfactant that can be degraded prior to MALDI. Another option would be to select a MALDI matrix (this chart can help you get an idea of what is out there) that helps offset the suppression of ionization due to a surfactant. We generally use MBT with surfactants, and it works really well with low levels of SDS for example, although I haven't tried it with Tween20.

What matrix are you currently using? I have found that some matrices are incompatible with a reconstitution solution that contains phosphate, from my experience 50% acetonitrile works as a good all around "go to" reconstitution solution.

Edited by proteaMatt, 28 March 2012 - 06:52 AM.

Lab Technician at Protea Biosciences




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