6 replies to this topic

[Juliasarmoire]

I'm still trying to finish my master's thesis... and my next big step is doing a dialysis. I have a protein fragment, about 37.6 kDA, with histidine tags which has been purified (more or less). As my protein formed inclusion bodies, I used 8M urea, meaning my protein is in an elution buffer containing 8M urea, 250mM imidazole, 50mM NaH2PO4 and 300mM NaCl (Protino Ni-TED columns). I have tried a dialysis once before with a small amount of protein, just to see how it goes... my protein seemed to be gone (more or less). It works with Western Blotting, but the concentration is too low even for silver staining. I used a Slide-A-Lyzer Dialysis Cassette, which seemed basically easy to use.

I dialysed my sample against 4M urea overnight (50mM NaH2PO4 and 300mM NaCl, but NO imidazole!)
then 12 hours against 2M urea (50mM NaH2PO4 and 300mM NaCl, but NO imidazole!)
and finally against PBS for another overnight (buffer was changed three times, but not before this test, while saving urea! sometimes playing a scientist is hard in the lab when everything is supposed to cost too much )

my big question is that do I need to add imidazole for my dialysis buffer too?
I couldn't see any protein precipitations, but maybe they were too small...
boss is saying that getting the protein to 2M urea should be enough for a rabbit injection...

but is there any magical tricks how to do the dialysis right at once?
also the cassette volume was supposed to be between 12-30ml, but mine was only 5ml... can it have any impact for the outcome?

Thanks

[ProteinWork]

Juliasarmoire, on Apr 19 2010, 03:56 PM, said:

I'm still trying to finish my master's thesis... and my next big step is doing a dialysis. I have a protein fragment, about 37.6 kDA, with histidine tags which has been purified (more or less). As my protein formed inclusion bodies, I used 8M urea, meaning my protein is in an elution buffer containing 8M urea, 250mM imidazole, 50mM NaH2PO4 and 300mM NaCl (Protino Ni-TED columns). I have tried a dialysis once before with a small amount of protein, just to see how it goes... my protein seemed to be gone (more or less). It works with Western Blotting, but the concentration is too low even for silver staining. I used a Slide-A-Lyzer Dialysis Cassette, which seemed basically easy to use.

I dialysed my sample against 4M urea overnight (50mM NaH2PO4 and 300mM NaCl, but NO imidazole!)
then 12 hours against 2M urea (50mM NaH2PO4 and 300mM NaCl, but NO imidazole!)
and finally against PBS for another overnight (buffer was changed three times, but not before this test, while saving urea! sometimes playing a scientist is hard in the lab when everything is supposed to cost too much )

my big question is that do I need to add imidazole for my dialysis buffer too?
I couldn't see any protein precipitations, but maybe they were too small...
boss is saying that getting the protein to 2M urea should be enough for a rabbit injection...

but is there any magical tricks how to do the dialysis right at once?
also the cassette volume was supposed to be between 12-30ml, but mine was only 5ml... can it have any impact for the outcome?

Thanks

Try using a buffer exchange column instead of dialysis. It's fast and easy, and your protein will not get dilute because of dialysis. I've used buffer exchange columns to process my protein before biotin-labeling the protein, it works as well as the overnight dialyzed protein sample.

[mdfenko]

ProteinWork, on Apr 19 2010, 05:08 PM, said:

Try using a buffer exchange column instead of dialysis. It's fast and easy, and your protein will not get dilute because of dialysis. I've used buffer exchange columns to process my protein before biotin-labeling the protein, it works as well as the overnight dialyzed protein sample.

not a good idea. the protein will probably precipitate on the column. try dialyzing in smaller steps of urea (you don't need to go overnight for each step, 3 hours should be okay for each step).

make sure you flush the membrane when you remove the protein.

using too large a dialysis cassette will lead to increased volume of your sample.

[paramyosin]

As far as you maintain your protein in low concentration, you probably will save it from aggregation when you are eliminating urea. I don´t know which is your idea, but i have used for rabbit inoculation even more urea than 2 M (8 M). You will obtain the immunity answer just against lineal epitopes but....sometimes is enough. If your idea is perform a concentration of the protein just after the dialysis i would let some urea in the protein solution, if the protein is unstable, i think it would go aggregation when you try to concentrate it.

[ProteinWork]

mdfenko, on Apr 23 2010, 04:01 PM, said:

ProteinWork, on Apr 19 2010, 05:08 PM, said:

Try using a buffer exchange column instead of dialysis. It's fast and easy, and your protein will not get dilute because of dialysis. I've used buffer exchange columns to process my protein before biotin-labeling the protein, it works as well as the overnight dialyzed protein sample.

not a good idea. the protein will probably precipitate on the column. try dialyzing in smaller steps of urea (you don't need to go overnight for each step, 3 hours should be okay for each step).

make sure you flush the membrane when you remove the protein.

using too large a dialysis cassette will lead to increased volume of your sample.

I have mostly worked on small protein/peptides which usually do not have precipitation problems in most buffer. I've also tried human alpha thrombin which is around 36kD and had observed no precipitation in the column. But the proteins I worked with do not require urea for solubility. So I agree that Juliasarmoire might be better off with step dialysis.

[AllenChiu]

I noticed that you mentioned to add imidazole to prevent his-tagged protein from aggregation.
Are aggregation supposed to happen without imidazole?
What bothered me is the Ni beads became very viscous/sticky after incubating with E. coli lysis of His tagged protein.
I don't know if it is because the sonicated E. coli lysis is too cencontrated (100ml medium of E. coli sonicated in 7ml buffer) or because there is any step else that I haven't realized.
What do you think?

[NHI]

hi fren..

well i think among options that u may try is

1) VivaSpin at 10kDa MWCO...u can check any brand...sartorius, millipore..etc
2) Use desalting column(Gel Filtration)....u can check thru GE chromatography resin...or any other brand

this is from my own experience...tq