Precipitation of peptide from a large volume - (Jun/13/2007 )
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?
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
They say you can run 100mL over 30mg/1mL column so I think if you use large enough column everything should be fine.
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).
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.
Yes, as good as I know it's "standard" method.