How can a protein without a G-site bind to GSH?

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    • #17024
      hurzz
      Participant

      Hello

      can someone help me understand my situation? i am new at this.

      I used GSH-Sepharose 4B beads on a cell lysate and got a single protein band on SDS-PAGE. I sent it for MALDI-TOF, the sequence came back with 3% coverage that has homolog with acetyl carrier protein.

      question:
      1. how can my protein bind to GSH without having a G-site?
      2. is it a case of bad prep?

      Thank you.

    • #112948
      JackBean
      Participant

      3% coverage is not very high, what was your score? That was the only detected protein?

    • #113030
      hurzz
      Participant

      Hello Mr JackBean

      yes i only got 1 band and the score for that 3% sequence is 100% of an acyl carrier protein lpxA-like motive.
      my suspicion is this animal utilises an analog of GSH.

      Thank you very much.

    • #113031
      JackBean
      Participant

      Can you post or PM me the sequence?

    • #113033
      hurzz
      Participant

      i have PM u. Thank you very much.

    • #113034
      hurzz
      Participant

      I re-post this, just in case you didn’t receive my PM. Thank you for your time.

      1 MIDETAYIHP SAIVEDGAVI GANVRIGPFC CIGSQVEIGE GTELKSHVVV NGITKIGRDN QIFQFASIGE MNQDLKYHGE
      81 PTRVEIGDRN RIRESVTIHR GTVQGGGVTK IGNDNLLMVN AHIAHDCIVG DRCVIANNGT LGGHVILGDY VIIGGMSAIH
      161 QFCQIGSHAM VGGCSGVVQD IPPYVIAQGN HATPFGTNVE GLKRRGFDKD SLNVIRNAYK ILYRNGKTLE EAQQEIAELA
      241 ENNQHVKIFS DFLANSTRGI VR

      the sequence IFS DFLAN is the homologue.

    • #113058
      JackBean
      Participant

      This is the protein which was identified, right? But only 3% of it was identified by MALDI-TOF, right? In such case you can have other protein, which is not in your database.

    • #113059
      JackBean
      Participant

      Is your species really Photorhabdus luminescens?

    • #113064
      hurzz
      Participant

      yes 3% by maldi-tof. my species is horseshoe crab. i suspect the protein is not in database. do you think i should proceed with N-terminal sequence for whole protein sequence then?

    • #113065
      hurzz
      Participant

      species the sample from is Tachypleus gigas.

    • #113066
      JackBean
      Participant

      Really? That’s interesting :o)

      Anyway, back to the problem. If you have 3% from 300 AA protein, that is only 6 amino acids, right? You cannot use that for identification. I bet that even the size doesn’t fit.
      Try the N-term. sequencing, but that will not help that much. If you have enough of protein (and it’s sufficiently clean), they should be able to sequence it using the MALDI-TOF.
      In any case, because you will probably not have the protein in any database, you will have to create some gDNA/cDNA library and use blotting with (degenerated) probe.

    • #113074
      hurzz
      Participant

      Thank you very much, pretty much what i thought as well.

      i have to find a facility where i can do N-term sequencing. we don’t have it here.

      i have abt 1.5mg of protein, ran on SDS-PAGE got a single band, but when i did 2DE i got 2 spots of the same MW but different pI. sent it for MALDI-ToF-ToF, this is the result i got. I was hoping that this protein would be a new protein.

      I just wanted assurance to rule out the possibility of this being a preparation error.

      Thank you for your input.

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