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    • #16272
      Valya
      Participant

      Hello,
      I have problem with cleaving a peptide (3,2 kDa) from 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.

    • #110353
      jonmoulton
      Participant

      Have you tried flying the peptide alone in the MALDI MS as a positive control?

      You might see sodium association with the peptide in the MALDI shifting the peak by the mass of sodium. Likely you’d see the peak with and without sodium.

    • #110354
      JackBean
      Participant

      You have lot’s of non-volatile substances, which may cause troubles in mass spec. But I’m not expert in this area.

      Do you have linear or reflected TOF? Because you could be at the limit of your machine with 3 kDa.

      Anyway, I would rather focus on the large protein, that should be easier to see both on gel and on MS.

    • #110356
      Valya
      Participant

      re: jonmoulton
      One person before me visulised it, but her probe didn’t contain so much side substances.. My spectrum is so bad that I cant see there any peak. It’s unpossible that the probe is empty, so that I have to decide what to eliminate.. I think about trying to cleave the pure probe without buffering agents..

      re: JackBean
      Reflected one. Actually if to discuss the big protein, I can see the band below the band of whole protein on SDS gel. So the result is positive, but doubtful because other nonspecific bands appear too..=\

    • #110361
      JackBean
      Participant

      Then try to cut the bands from gel and identify on MALDI or pepsin digest/LC-MS

    • #110374
      Valya
      Participant
      quote JackBean:

      Then try to cut the bands from gel and identify on MALDI or pepsin digest/LC-MS

      Allready in progress 🙂 Actually I have found a good question about tricine gel problem in Schägger protocol.. Will try it too.

    • #110395
      JackBean
      Participant

      what question?

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