My protein getting trapped during FPLC
dk at no.email.thankstospam.net
Tue Nov 30 20:57:48 EST 2004
On 30 Nov 2004 21:04:35 -0000, nikolai.scherbak at nat.oru.se (Nikolai Scherbak)
>I am trying to purify a protein using ion exchange chromatography column
>Resource Q connected to Akta Explorer 10. Protein has His-tag and FPLC is a
>second step of purification after IMAC.
>It is looks like my protein is getting trapped in IEC column (or just
>before) and elutes in a relatively small amount after approx. 0,4 M NaCl.
>Nothing comes out during sample loading on column and nothing elutes after
>wash of the column by of 1M of NaCl, 5 CV .
>After cleaning with 1M NaOH I am getting a top that is up to 20 times higher
>then what I am getting after elution with the buffer with NaCl.
>PI of my protein is 5.3 and I am using 20mM Tris-HCl buffer, pH 8.0.
>I would appreciate very much any help or explanation for this situation.
You are dealing with the not-to-unusual case of low recovery from the column.
Stuff happens. Read any comprehensive book on chromatography (the one
by Lev Osterman "Chromatograpy of Proteins and Nucleic Acids is particularly
recommended) for the detailed explanations. Briefly:
Mosat likely your protein interacts too strongly with the column. Either there
is too much hydrophibic interaction to begin with or there is too high a charge
density on the matrix that the protein gets "crucified" by too many contacts
with the insoluble support and denatures as a result. And denatured protein(s)
tend to become insoluble and stick everywhere like hell.
Both of the scenarios are quite likely in case of the [relatively] hydrophonbic
and VERY charged Resource columns. IMHO, your best bets if you insist
going IEC route:
a) bind your protein in moderate salt so that the initial binding strenght is
not too high; 150 mM salt maybe?
b) Change pH of your IEC to where yout protein is considerably less acidic;
pH 6.8 buffered with imidazole may be a good starting point?
c) Coupled or not with the #1 above - switching to a martrix that has variable
charge density. A classic DEAE is a good choice but if you insist on "high
performance" sorbents, I think Bio-Rad and Toyo have some metacrylate-based
weak ion-exchangers. There, merely my changing pH you can reduce the
capacity of the matrix for your protein and - maybe - increase recovery.
d) Increase concentration of "stabilizers" that also reduce non-specific
interactions. 10-20% glycerol or ethylene glycol is typical (dodn't forget to
lower flow rate!).
Best of luck,
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