[Fwd: FITC unconjugated]

From: Kevin Waddick (waddi002@tc.umn.edu)
Date: Mon Aug 09 1999 - 09:23:26 EST 

Kevin Waddick wrote:

>     I am referring to the messages shown below and am wondering what happens
> when FITC "reacts with water quite rapidly." Can it no longer be conjugated to
> a protein, can it no longer be excited to fluoresce, or what? If it can no
> longer be covalently conjugated to a protein, can it still be used
> unconjugated to bind proteins in order to measure total cellular protein
> content? Lastly, when FITC is said to be stable for hours rather than days in
> DMSO, does this mean at RT, 4 C, -20 C, or -70 C? I have used FITC stored for
> years in DMSO at -20 C in order to measure intracellular protein content and,
> while it seemed to do what it was supposed to when analyzed by FCM, the
> results were not as clear as I expected from looking at figures in
> publications.
>    I would appreciate a little more information on this topic.
>
> Dr. Kevin G. Waddick
> Hughes Institute
>
> Mario Roederer wrote:
>
> > >Hello,
> > >Does anyone have any experience with the storage of unconjugated FITC
> > >(isomer 1, Sigma, F7250) ? I have dissolved it in PBS, pH8, 0.1mg/ml
> > >and I keep it in the dark at 3-4°C.  Does anyone know something about
> > >the stability in this form and how long it can be stored like this ?
> > >
> > >Thanks
> > >Isabel
> >
> > About 30 minutes.  FITC reacts with water quite rapidly...  never store it
> > in water!
> >
> > Since the cost of FITC is so low compared to the cost of everything else
> > associated with the reaction, you should always use a freshly-opened vial
> > when you are about to use it; discard the rest.  Also, dissolve the FITC in
> > DMSO and then add it to your reaction.  Make sure that your DMSO is the
> > highest-grade possible, and keep the DMSO in a dessicator to make sure it
> > has no water.
> >
> > Note that you cannot use pipetteman to accurately pipet DMSO--you have to
> > use glass (not plastic!) pipettes.  Typically, we break the ampule, and add
> > 1.0 mL of DMSO from a glass pipet.  This results in 10mg/ml solution, which
> > is great for most reactions.  (Then you will have to add the small amount
> > you need using a pipettman; using yellow tips works reasonably well but you
> > have to operate quickly:  the DMSO does not hold well inside the tip and
> > leaks out).
> >
> > FITC in DMSO is relatively stable (i.e., hours... not days), when kept
> > unexposed to air & water.
> >
> > mr
> >
> > -- End --

attached mail follows:

I am referring to the messages shown below and am wondering what happens when FITC "reacts with water quite rapidly." Can it no longer be conjugated to a protein, can it no longer be excited to fluoresce, or what? If it can no longer be covalently conjugated to a protein, can it still be used unconjugated to bind proteins in order to measure total cellular protein content? Lastly, when FITC is said to be stable for hours rather than days in DMSO, does this mean at RT, 4 C, -20 C, or -70 C? I have used FITC stored for years in DMSO at -20 C in order to measure intracellular protein content and, while it seemed to do what it was supposed to when analyzed by FCM, the results were not as clear as I expected from looking at figures in publications. I would appreciate a little more information on this topic. Dr. Kevin G. Waddick Hughes Institute Mario Roederer wrote: > >Hello, > >Does anyone have any experience with the storage of unconjugated FITC > >(isomer 1, Sigma, F7250) ? I have dissolved it in PBS, pH8, 0.1mg/ml > >and I keep it in the dark at 3-4°C. Does anyone know something about > >the stability in this form and how long it can be stored like this ? > > > >Thanks > >Isabel > > About 30 minutes. FITC reacts with water quite rapidly... never store it > in water! > > Since the cost of FITC is so low compared to the cost of everything else > associated with the reaction, you should always use a freshly-opened vial > when you are about to use it; discard the rest. Also, dissolve the FITC in > DMSO and then add it to your reaction. Make sure that your DMSO is the > highest-grade possible, and keep the DMSO in a dessicator to make sure it > has no water. > > Note that you cannot use pipetteman to accurately pipet DMSO--you have to > use glass (not plastic!) pipettes. Typically, we break the ampule, and add > 1.0 mL of DMSO from a glass pipet. This results in 10mg/ml solution, which > is great for most reactions. (Then you will have to add the small amount > you need using a pipettman; using yellow tips works reasonably well but you > have to operate quickly: the DMSO does not hold well inside the tip and > leaks out). > > FITC in DMSO is relatively stable (i.e., hours... not days), when kept > unexposed to air & water. > > mr > > -- End --

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