c*****e
发帖数: 436 | 1 have to do long experiments with temperature ramping from -10C to 45C and
the whole experiments last for 2 weeks, on peptide in solution sample.
question: does this sample has to be prepared with sterilization?
Thanks!!!
Baozi will be given to helpful replies!!! |
m**z
发帖数: 787 | 2 i guess it really depends on how stable the peptide is... This is just like
proteins. I know proteins to be stable at 40C for days, other proteins can
only last for several hours. varying the solution conditions may help, but
this has to be tested...
I don't think sterilization is that important. Sterilization by a 0.2uM
filter is also easy to do though. But you may lose some sample during
fitration.
【在 c*****e 的大作中提到】
: have to do long experiments with temperature ramping from -10C to 45C and
: the whole experiments last for 2 weeks, on peptide in solution sample.
: question: does this sample has to be prepared with sterilization?
: Thanks!!!
: Baozi will be given to helpful replies!!!
|
c*****e
发帖数: 436 | 3 how to do sterilization, it seem every step has to be under a hood or
something? I've no knowledge of these stuff at all because I'm not biology
major.
my peptide is (Val-Pro-Gly-Val-Gly)3 lyophilized powder. Previously I just
add distilled water into it. And I use this sample at body temperature for
month. Does this mean all my work are garbage now?
like
【在 m**z 的大作中提到】
: i guess it really depends on how stable the peptide is... This is just like
: proteins. I know proteins to be stable at 40C for days, other proteins can
: only last for several hours. varying the solution conditions may help, but
: this has to be tested...
: I don't think sterilization is that important. Sterilization by a 0.2uM
: filter is also easy to do though. But you may lose some sample during
: fitration.
|
m**z
发帖数: 787 | 4 For your sample, 0.2uM filter will be fine. Don't know what kind of
containers you are using... But I still don't think it is that important...
I don't have much experience with small peptides.For proteins, buffer with
salt will usually be better than just water. Adding some detergent may also
help if you don't think it will interfere with your experiment. Also, note
that there may be TFA remaining in the lyophilized powder. So just adding
water will result in an acidic solution.
The only way I can think of to check the integrity of this peptide is by
mass-spec. It may tell you whether or not it is degraded. No idea about
whether your data is OK or not with the info you provided...
【在 c*****e 的大作中提到】
: how to do sterilization, it seem every step has to be under a hood or
: something? I've no knowledge of these stuff at all because I'm not biology
: major.
: my peptide is (Val-Pro-Gly-Val-Gly)3 lyophilized powder. Previously I just
: add distilled water into it. And I use this sample at body temperature for
: month. Does this mean all my work are garbage now?
:
: like
|
K******S
发帖数: 10109 | 5 don't worry, peptides are VERY stable. especially your peptide, I can't
think of any proteolytic enzyme that can cleave the sequence.
If you can, just make some aliquots
【在 c*****e 的大作中提到】
: how to do sterilization, it seem every step has to be under a hood or
: something? I've no knowledge of these stuff at all because I'm not biology
: major.
: my peptide is (Val-Pro-Gly-Val-Gly)3 lyophilized powder. Previously I just
: add distilled water into it. And I use this sample at body temperature for
: month. Does this mean all my work are garbage now?
:
: like
|
c*****e
发帖数: 436 | 6 thank you so much! what is aliquots, by the way?
【在 K******S 的大作中提到】
: don't worry, peptides are VERY stable. especially your peptide, I can't
: think of any proteolytic enzyme that can cleave the sequence.
: If you can, just make some aliquots
|
c*****e
发帖数: 436 | 7 it seems the Chemist synthesized this peptide told me that in this peptide
valine or some thing? can be oxidized? (don't know what does this mean).
And I saw from papers other people make similar sample with very complicated
steps with:
"Samples were prepared for the NMR experiments in 6-mm tubes by dissolving 0
.3g of lyophilized material in 1.5ml of distilled H2O. ..The tubes were
placed in a bath at 20C, and argon gas was passed through the samples for 6h
by means of long-tipped pipets placed into the bottom of the tubes. The
tubes were capped under argon atmosphere and incubated at 30C in a bath for
3 days, during which time the polypentapeptide had formed a coacervate in
the lower 4 cm of each tube. The equilibrium solution above the coacervates
was removed at 30C, and the tubes were purged with argon and recapped. .."
I have a few questions:
1. what is the purpose of that argon gas flow? is it to prevent oxidation?
or kill aerobic bacterias?
2. what is the coacervate? what's it like?why it need to incubate in bath
for 3 days? why need to remove equilibrium solution above it? can the
interface be seen by naked eyes?
3. it seems he didn't sterilize the sample, did he?
Thanks, anyone give me answer got baozi!
【在 K******S 的大作中提到】
: don't worry, peptides are VERY stable. especially your peptide, I can't
: think of any proteolytic enzyme that can cleave the sequence.
: If you can, just make some aliquots
|
m**z
发帖数: 787 | 8 The Val in your peptide won't get oxidized... Argon is for anaerobic purpose
. But since your peptide doesn't have Cys, don't think it is necessary. Don'
t know about your question 2... They didn't sterile as what I can tell from
their description.
For NMR, it should be easy to check your sample. Run a simple HSQC at the
end of your experiment to compare with the spectrum taken at the beginning.
This should give you a pretty good idea of your sample integrity.A little
precipitation during NMR may not interfere with your results.
complicated
0
6h
for
【在 c*****e 的大作中提到】
: it seems the Chemist synthesized this peptide told me that in this peptide
: valine or some thing? can be oxidized? (don't know what does this mean).
: And I saw from papers other people make similar sample with very complicated
: steps with:
: "Samples were prepared for the NMR experiments in 6-mm tubes by dissolving 0
: .3g of lyophilized material in 1.5ml of distilled H2O. ..The tubes were
: placed in a bath at 20C, and argon gas was passed through the samples for 6h
: by means of long-tipped pipets placed into the bottom of the tubes. The
: tubes were capped under argon atmosphere and incubated at 30C in a bath for
: 3 days, during which time the polypentapeptide had formed a coacervate in
|
c*****e
发帖数: 436 | 9 Argon for anaerobic purpose is not for sterilization? then for what?
oxidization? which residue?
Thanks for the HSQC suggestion, though I'm not able to do this now I'll
consider.
purpose
Don'
from
.
【在 m**z 的大作中提到】
: The Val in your peptide won't get oxidized... Argon is for anaerobic purpose
: . But since your peptide doesn't have Cys, don't think it is necessary. Don'
: t know about your question 2... They didn't sterile as what I can tell from
: their description.
: For NMR, it should be easy to check your sample. Run a simple HSQC at the
: end of your experiment to compare with the spectrum taken at the beginning.
: This should give you a pretty good idea of your sample integrity.A little
: precipitation during NMR may not interfere with your results.
:
: complicated
|
m**z
发帖数: 787 | 10 Argon bubbling is a method to create anaerobic condition (pseudo- as I will
say). Usually to prevent oxidation of Cys residues.
【在 c*****e 的大作中提到】
: Argon for anaerobic purpose is not for sterilization? then for what?
: oxidization? which residue?
: Thanks for the HSQC suggestion, though I'm not able to do this now I'll
: consider.
:
: purpose
: Don'
: from
: .
|
c*****e
发帖数: 436 | 11 thanks!
will
【在 m**z 的大作中提到】
: Argon bubbling is a method to create anaerobic condition (pseudo- as I will
: say). Usually to prevent oxidation of Cys residues.
|
