Protein precipitation during dialysis - (Aug/22/2007 )
Hi,
I purified His tagged protein size 48kD and pI 6.8 using Novagen protocol TB054. Protein was eluted in 20mM Tris, pH 7.9, 500 mm NaCl and 1M imidazole. Another protein, size 32kD was eluted in same buffer but with 250 mM imidazole. I started dialysing in 20 mm Tris, pH 8.0 and 50 mM NaCl at 4 oC in cold room. Protein precipitates within 45 minutes in the dialysis bag. Any suggestion.
Anjou
why are you dialyzing? what is the next step? if it is for gel filtration then you need higher salt to prevent nonspecific binding to the matrix.
you may require higher salt to maintain suspension.
you may require higher salt to maintain suspension.
In one of the proteins, I have to remove His tag using Enterokinase and use the protein for antibody production. Enterokinase does not work in the presence of imidazole. Other protein for protein-protein interaction.
can you try dialyzing with buffer without imidazole but everything else the same?
it just occurred to me, did you adjust the pH of your buffer before or after adding the imidazole? in my experience, people forget that imidazole is also used as a buffer and if it is not adjusted to the working pH will change the pH of your buffer. the pH change during dialysis may be causing your problem.
it just occurred to me, did you adjust the pH of your buffer before or after adding the imidazole? in my experience, people forget that imidazole is also used as a buffer and if it is not adjusted to the working pH will change the pH of your buffer. the pH change during dialysis may be causing your problem.
Yes, I did adjust the pH after adding imidazole.
I can try using the same buffer but without imidazole. But NaCl will be high after dialysis. I also read on the internet that removal of imidazole from His tagged proteins causes aggregation of protein. Whether this aggregation affects the activity of protein?
will high nacl have any effect on what you plan to do next?
aggregation often has a negative effect on enzyme activity. it can have an effect on conformation and/or conformational changes (caused by substrate binding). it can also block the active site. however, i have worked with some proteins which maintain activity when slightly aggregated.
Thanks for your suggestions. I have to use one protein for antibody production and other for protein-protein interaction.
I lose protein during dialysis. This time I started with 15 mg protein before dialysis and after dialysis and lyophilization. I had only 1 mg left. I was getting agreegates during dialysis which dissolves in 20mM Tris (pH 8.0) with 50 mM Nacl. Please suggest what to do.
Anju
you may require higher salt to maintain suspension.
In one of the proteins, I have to remove His tag using Enterokinase and use the protein for antibody production. Enterokinase does not work in the presence of imidazole. Other protein for protein-protein interaction.
I had the same problem that you are now experiencing. In the end I couldnt dialyse the imidazole out without the protein preciptating.
I solved the issue by using the precipitate for antigen injection into the animals anyway. Apparently in some cases the precipitated protein can cause a larger immuno response due to the inability of the host to remove the antigen in the same timely fashion as a soluble antigen.
In addition, I found the adjuvant-antigen mix to be "thicker" and easier to form than when doing it with a soluble antigen.
I was worried for a while that the HIS tag would cause the titre to contain anti-his antibodies...but it didnt (well not that much that it detects non-specific His signals) and I now have three different antibodies prepared this way with great titre & specificity.
Just remember that you are still required to dialyse the imidazole out for ethics reasons for the animal and have the antigen preparation in a suitable solution i.e PBS.