Hello everyone.
I explain my case: I had a protein with this purification protocol: Histrap-MonoQ-Superdex. The protein buffer is Tris 20mM 150mM NaCl pH:7.4. In this case, the protein degraded or autoproteolized after 2 weeks.
Now, We are using a new purification protocol without MonoQ ( becuase it isnt necesary) and now the protein degrades or autoproteolize slower ( It has been kept 2 weeks and the degradation or autoproteolysis is minimal)
So I only change the MonoQ step, which first I diluated the protein until 20mM NaCl and them in the column the protetein eluated at 300mM NaCl ,I keep the samples at 4ºC Overnight and the next day I did the gel filtration and stored it with the protein buffer.
So my question is, if this MonoQ step could activated/increase the protelysis activity of the protein or increased its degradation?
It is strange because the final buffer in both protocols is the same, the only change is the MonoQ step.
Thank you
The higher salt concentration may increase the activity of a contaminating protease, or it may slightly denature your protein making it more susceptible to proteolytic degradation (NaCl is a mild chaotrope), or the MonoQ column may be contaminated with protease.
Avoid degradation of the purified protein by storing it frozen in small single-use aliquots.
Hello David, it is very possible that excess NaCl must have affected the stability of your protein. The fraction can be chromatographed by passage through a Mono Q anion-exchange column (HR 5/5) attached to a Pharmacia Fast Protein Liquid Chromatography (FPLC) system. The sample is clarified by centrifugation, loaded onto the column and eluted with a discontinuous gradient consisting of steps of 0 to 0.3 M, 0.3 to 44 M and 0.44 to 1.2 M NaCl, as an example. The fractions enriched in the target protein are pooled and centrifuged at 12 000 x g for 10 min to remove insoluble material. The supernatant is dialyzed for 4 to 6 h against 20 mM Tris-HCl, pH 7.5 to remove NaCl, brought to 80% (NH4)2SO4 and the precipitated fraction is resuspended in 20 mM Tris-HCl, pH 7.5. If necessary, residual salt can be removed by passage through a small gel filtration column. For applications requiring cation-exchange columns carboxymethyl (CM)-cellulose or CM-Sepharose can be employed. Just look up your protocol again. Pay attention to the dialysis step.
The higher salt concentration may increase the activity of a contaminating protease, or it may slightly denature your protein making it more susceptible to proteolytic degradation (NaCl is a mild chaotrope), or the MonoQ column may be contaminated with protease.
Avoid degradation of the purified protein by storing it frozen in small single-use aliquots.
NACl may slightly denature the protein that makes the protein more susceptible to proteolytic degradation.so, the higher salt concentration may increase the protease activity.
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