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

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

Postby hurzz » Mon Nov 12, 2012 12:34 pm

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.
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Postby JackBean » Mon Nov 12, 2012 3:01 pm

3% coverage is not very high, what was your score? That was the only detected protein?
http://www.biolib.cz/en/main/

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

Postby hurzz » Thu Nov 22, 2012 11:19 am

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.
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Postby JackBean » Thu Nov 22, 2012 12:07 pm

Can you post or PM me the sequence?
http://www.biolib.cz/en/main/

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Postby hurzz » Thu Nov 22, 2012 12:51 pm

i have PM u. Thank you very much.
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Postby hurzz » Thu Nov 22, 2012 5:52 pm

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.
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Postby JackBean » Sat Nov 24, 2012 8:58 pm

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.
http://www.biolib.cz/en/main/

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Postby JackBean » Sat Nov 24, 2012 8:59 pm

Is your species really Photorhabdus luminescens?
http://www.biolib.cz/en/main/

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Postby hurzz » Sun Nov 25, 2012 7:36 am

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?
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Postby hurzz » Sun Nov 25, 2012 7:37 am

species the sample from is Tachypleus gigas.
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Postby JackBean » Sun Nov 25, 2012 1:08 pm

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.
http://www.biolib.cz/en/main/

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

Postby hurzz » Mon Nov 26, 2012 3:40 am

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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