Dear Lalitha

Generally i perform protein purification with resind packed on coloumns also for small volumes and therefore i'm not able to observe if protein binding in some cases cam induce beads aggregation but i suspect that if the protein is not monomeric but oligomeric or aggregate, it can act as a crosslinker beetween beads and induce a similar phenomena. did you tried to go one with the purification and observe whats happen after elution? because if i'm right once you have extracted protein from the beads with elution buffer the beads will return at the original state ( no aggregation)

is your protein contains

many cysteines or other metrl esidues that can find the cobalt? because also this properties may justify it ability to act as crosslinker beetween the beads.

good luck

Manuele

Like Manuele, I always pack the beads into a column of a suitable size: I connect the outflow to a flow-through UV monitor and chart recorder. After sample application I wash to baseline then elute the protein.

Aggregation might be due to any of the compnents in a crude cell extract - for example DNA if your protein is DNA binding.

Is there still aggregation after washing the beads free of "unbound" material?

I use 50 mM phosphate buffer pH 8.0 without NaCl, but the elution buffer is 50 mM phosphate buffer pH 7 and 500 mM NaCl. I think that you need to decrease the concentration of NaCl and do it at 4 C.

Dear Geoff Margison

You made a very interesting point. Yes, my protein is DNA binding. Do you think adding DNase to the crude extract will help me solve this issue if the aggregation is due to DNA binding?

And to answer your question, yes. The aggregation persists to some extent even after washing away the unbound materials. But the curious thing is that even though the beds aggregate to a great extent, the protein remains almost unbound to the column. Most of my expressed protein washes out in the flowthrough. Can you please give me the name and make of the column you use for His-tag purification?

Thanks a lot,

Lalitha

Dear Manuele Martinelli ,

I did proceed with the purification, but found that most of my protein is not binding to the column at all. It is mostly washed away with the flowthrough or the subsequent washes. Only a fraction of the protein was obtained after the final elution. Even though the beads get less aggregated, they do not return to their original state after elution. Can you tell me the name and make of the beads and column you use for His-tag purification?

Thanks a lot,

Lalitha

Dear Lalitha

generally as beads i'm using Nichel sepharose FF (GE) and i pack it by gravity on Empty pd10 coloumn (form 1 to 3 ml of beads) or in a Bio spin coloum (biorad) for small scale trial a(100-200 il of resin)

if your protein was away into the flow through also with this approach it mean a that it is probably aggregates and the tag is not well exposed and robably you have to optimize:

- expression condiction

- buffer composition

- or protein domain

good luck

Manuele

https://www.sigmaaldrich.com/catalog/product/roche/cohisrro?lang=en®ion=GB&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold5-4

I pack this into a suitable sized plastic syringe into which I have inserted a plastic sintered plug, but premade columns are available for self-packing.

I dont know how you prepare your extracts, but I use sonication (which yields average ~400bp fragments) , which may reduce any DNA binding problems. I guess that addition of DNAse would be even better, but I never tried it. In addition, I do not add any imidazole to the extraction buffer. I had planned to re-assess this at a later stage, but as my protein was active and sufficiently pure for my purposes, I didnt bother!

Sorry i forgot to attach a file that can help you in the protocol definition.

it is a protocol for the purification for mammalian cell surnatant but the e.coli differ only for the addiction of cell lisys step ( sonication or chemical/enzimatic)

good luck

Manuele