Dear all,
recently, i prepared the ATP solution used to assay ATPase activity. when the
Control Well just containing assay buffer and ATP mixed with molybdate ,the
colour changed to green, this mean that there is free pi in the
solution.the assay buffer and water is free from pi.and the ATP
The commonest error with averaging is getting the mask wrong.
Check that the CCs after application of the averaging start at a
reasonable value - 0.3 at least and increase with each cycle ( by the
way why do ncycle 1?)
But in the end the density will not be identical, the Fobs are not
Hello one and all !!
I have been working on cloning and purification aspects on a Leishmanial
cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX
expression vector. Expression seemed quite okay when induced with 0.5 and
1mM IPTG, so did the solubility in 4 buffers at
Hello one and all !!
I have been working on cloning and purification aspects on a Leishmanial
cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX
expression vector. Expression seemed quite okay when induced with 0.5 and 1mM
IPTG, so did the solubility in 4 buffers at
It sounds like list your GST construct is not binding to the column
(or very well) when the peptidase is attached. GST needs to form a dimer
to binding to the column - I suspect that your construct interferes with
dimer formation - when the peptidase is present, but when not there due
to
I worked with several viral cystene proteases. I do not recommend using
basic pH for them dring purification or storage or crystallization. Keep it
below 7, because the active site cysteine oxydazes very easily. The higher
the pH, the easier the oxydation. PH 10 can also cause hydrolysis of
Stability of ATP (to hydrolysis) is pH dependent, and even the disodium ATP
is strongly acidic in distilled water. I used to make 100 mM ATP by
dissolving disodium or Na,K-ATP in ~100 mM NaOH, getting pH ~6-7.
This suggests 8.3 would be better:
Hello,
I'm trying to use the 'probe clashes' function in WinCoot. However, each
time I try I get the following error message:
Found 4580 hydrogens 0 hets
Standardized 6012 hydrogens 0 hets
Added 344 hydrogens 0 hets
Removed 0 hydrogens 0 hets
Adjusted 123 groups(s)
If you publish work which uses
Hi,
Thanks for reminding me checking the mask. I think their might be
something wrong with the mask, since when DM read in the mask, it says:
Number of columns, rows, sections ... 84 74 69
Map mode 0
If you are using malachite green assay, there will be an issue with the
solution turning green at high ATP concentrations. There is a protocol to
reduce ATP hydrolysis after the addition of malachite green
(http://www.ncbi.nlm.nih.gov/pubmed/16674910). An alternative to malachite
green assay
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