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Precipitation of peptide from a large volume - (Jun/13/2007 )

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Ok, that's similar to what I've been reading. Thanks. Can I run as much volume over it as I want? For collection of peptide that is present in low quantities, I need to run a reasonably large volume - perhaps 600mL. I realise that the larger the volume, the more contaminants I'm likely to collect... but better that that ending up with no peptide of interest. I'll worry about removing contaminants once I have something to work with! I expect that I'll be able to put the sample from this column through an HPLC with a similar column but elution with a solvent gradient to separate out what I collect on these kindns of columns with a complete elution?

-ScottC-

If I understand truly , your peptide solution may contain other peptides contaminants. So do SPE with total elution at high ACCN %, then concentrate and evaporate ACCN on rotor evaporator and apply on RPC8 column ( for ex Xterra ( 4,6*150) Waters) then elute in ACCN gradient ( 60min 1 ml\min for the first elution to analyse profile)
with 0.1% TFA, or if your peptides high acidic so use 50mM triethylamine in HPLC buffer as alternative ion pair agent

-circlepoint-

QUOTE (ScottC @ Jun 15 2007, 03:22 PM)
Can I run as much volume over it as I want? For collection of peptide that is present in low quantities, I need to run a reasonably large volume - perhaps 600mL.

They say you can run 100mL over 30mg/1mL column so I think if you use large enough column everything should be fine.

QUOTE (ScottC @ Jun 15 2007, 03:22 PM)
I realise that the larger the volume, the more contaminants I'm likely to collect... but better that that ending up with no peptide of interest. I'll worry about removing contaminants once I have something to work with! I expect that I'll be able to put the sample from this column through an HPLC with a similar column but elution with a solvent gradient to separate out what I collect on these kindns of columns with a complete elution?

That's right. You can also do that before HPLC by optimizing SPE protocol (eg. change AcCN concentration for wash and elution) and if your contaminants are >15kDa you can use proper column to exclude them (eg. Phenomenex Strata C18-E or Strata X).

-K.B.-

QUOTE (K.B. @ Jun 14 2007, 05:39 PM)
Low-range MW marker from Sigma have 1060 Da band that is clearly visible on tris-tricine sds-page. My labmate managed to visualize peptides somewhere in range of 600-800 Da.


Is this with the 'standard' tris-tricine gels described by Schägger & von Jagow? Or does your labmate use a modified method for these small peptides? I'd be interested if you/your labmate would be willing to share the protocol.

Cheers,

Scott.

-ScottC-

Yes, as good as I know it's "standard" method.

-K.B.-

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