Phaloïdin - Alexa Fluor 647 conjugates: variability in labeling efficiency?

[lechristophe]

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Dear microscopists,

We are extensively using phalloidin conjugated to Alexa Fluor 647 (from Molecular Probes/Invitrogen/Thermo Fisher) to do STORM super-resolution microscopy of actin in various fixed cells. The smallest quantity sold is 300U, which are to be dissolved in 1.5 mL MeOH and stored at -20°C per manufacturer instructions.

We have noticed two things:

- When reconstituted and stored this way, the labeling quality seems to go down after a few months. We are using a high concentration of phallloïdin for labeling (1/12 i.e. 0.5 µM) overnight at 4°C, and the labeling efficiency is directly measurable by the overall intensity at diffraction-limited level, as well as the number of localizations we get when performing STORM. 

- When we saw this drop in quality, we ordered a new vial of phalloidin-AF647 from Thermo Fisher. We got the same lot number than our previous one, and labeling quality was quite bad from the start. We got it replaced at no charge by a newer lot number. This newer lot is a bit better, but the labeling is definitely not as good as with some of the previous lots.

I guess my questions are :

- Is it known that conjugated phalloidin (especially AF647) labeling efficiency can vary from lot to lot? Could this be related to purity, presence of unlabeled phalloidin, or am I on the wrong track here?

- Should we test several lots in parallel with a standardized labeling assay (but this would be really expensive given the 300U smallest sold vial)? 

- Once we find a suitable lot, is there an alternative way to reconstitute/store the phalloidin to ensure preservation over months? I was thinking drying-down smaller aliquots with a suitable solvent and speedvac, like we do for fluorophores NHS-esters, but I'm not sure what solvent to use.

I know there are people from Molecular Probes/Thermo Fisher on this list, so any input from them would be also very helpful. I've also noticed that phalloidin-AF647 is available from another supplier (Cytoskeleton Inc.), not sure if these have the same production source, or if people have seen differences between suppliers.

Thanks,

Christophe

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France

[Jason Miller]

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Hi Christophe-

 

As you probably know, methanol fixation destroys the phalloidin antigen. I wonder whether any of the carry-over methanol you are using to store your phalloidin is enough to tweak the antigen?

 

We have used phalloidin from Biotium without issue:

https://biotium.com/product/phalloidin-cf647-conjugate/

 

-Jason

 

 

-------

Jason Miller, MD, PhD

University of Michigan Kellogg Eye Center

 

Home address:

117 Worden Ave

Ann Arbor, MI 48103

Cell: [phone]

E-mail: [hidden email]

 

"What has once been published is usually spoken of as "known," and it is often forgotten that the rediscovery in the library may be a more difficult and uncertain process than the first discovery in the laboratory." -- Lord Rayleigh, 1884

 

On Sat, Oct 22, 2016 at 10:59 AM, Christophe Leterrier <[hidden email]> wrote:

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Dear microscopists,

 

We are extensively using phalloidin conjugated to Alexa Fluor 647 (from Molecular Probes/Invitrogen/Thermo Fisher) to do STORM super-resolution microscopy of actin in various fixed cells. The smallest quantity sold is 300U, which are to be dissolved in 1.5 mL MeOH and stored at -20°C per manufacturer instructions.

 

We have noticed two things:

- When reconstituted and stored this way, the labeling quality seems to go down after a few months. We are using a high concentration of phallloïdin for labeling (1/12 i.e. 0.5 µM) overnight at 4°C, and the labeling efficiency is directly measurable by the overall intensity at diffraction-limited level, as well as the number of localizations we get when performing STORM. 

 

- When we saw this drop in quality, we ordered a new vial of phalloidin-AF647 from Thermo Fisher. We got the same lot number than our previous one, and labeling quality was quite bad from the start. We got it replaced at no charge by a newer lot number. This newer lot is a bit better, but the labeling is definitely not as good as with some of the previous lots.

 

I guess my questions are :

- Is it known that conjugated phalloidin (especially AF647) labeling efficiency can vary from lot to lot? Could this be related to purity, presence of unlabeled phalloidin, or am I on the wrong track here?

- Should we test several lots in parallel with a standardized labeling assay (but this would be really expensive given the 300U smallest sold vial)? 

- Once we find a suitable lot, is there an alternative way to reconstitute/store the phalloidin to ensure preservation over months? I was thinking drying-down smaller aliquots with a suitable solvent and speedvac, like we do for fluorophores NHS-esters, but I'm not sure what solvent to use.

 

I know there are people from Molecular Probes/Thermo Fisher on this list, so any input from them would be also very helpful. I've also noticed that phalloidin-AF647 is available from another supplier (Cytoskeleton Inc.), not sure if these have the same production source, or if people have seen differences between suppliers.

 

Thanks,

 

Christophe

 

**********************************************************
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[lechristophe]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Jason,

The possibility that residual MeOH impairs labeling is a good point. When I started doing thses experiments I compared staining with phalloïdin 1/12 with or wihtout drying the MeOH before diluting it in phosphate buffer. It made no difference at the time but maybe I could revisit this with the new lots.

I have tried phalloïdin-CF647 from Biotum, but the staining was definitely not as dense and bright than with AF647. This is not critical for routine staining, but for STORM imaging we need the highest labeling density possible. By the way I made a mistake in my original post, the other supplier of phalloïdin-AF647 is not Cytoskeleton Inc, but Cell Signaling Technologies.

Christophe

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France

On Sat, Oct 22, 2016 at 7:07 PM, Miller, Jason <[hidden email]> wrote:

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Hi Christophe-

 

As you probably know, methanol fixation destroys the phalloidin antigen. I wonder whether any of the carry-over methanol you are using to store your phalloidin is enough to tweak the antigen?

 

We have used phalloidin from Biotium without issue:

https://biotium.com/product/phalloidin-cf647-conjugate/

 

-Jason

 

 

-------

Jason Miller, MD, PhD

University of Michigan Kellogg Eye Center

 

Home address:

117 Worden Ave

Ann Arbor, MI 48103

Cell: [phone]

E-mail: [hidden email]

 

"What has once been published is usually spoken of as "known," and it is often forgotten that the rediscovery in the library may be a more difficult and uncertain process than the first discovery in the laboratory." -- Lord Rayleigh, 1884

 

On Sat, Oct 22, 2016 at 10:59 AM, Christophe Leterrier <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Dear microscopists,

 

We are extensively using phalloidin conjugated to Alexa Fluor 647 (from Molecular Probes/Invitrogen/Thermo Fisher) to do STORM super-resolution microscopy of actin in various fixed cells. The smallest quantity sold is 300U, which are to be dissolved in 1.5 mL MeOH and stored at -20°C per manufacturer instructions.

 

We have noticed two things:

- When reconstituted and stored this way, the labeling quality seems to go down after a few months. We are using a high concentration of phallloïdin for labeling (1/12 i.e. 0.5 µM) overnight at 4°C, and the labeling efficiency is directly measurable by the overall intensity at diffraction-limited level, as well as the number of localizations we get when performing STORM. 

 

- When we saw this drop in quality, we ordered a new vial of phalloidin-AF647 from Thermo Fisher. We got the same lot number than our previous one, and labeling quality was quite bad from the start. We got it replaced at no charge by a newer lot number. This newer lot is a bit better, but the labeling is definitely not as good as with some of the previous lots.

 

I guess my questions are :

- Is it known that conjugated phalloidin (especially AF647) labeling efficiency can vary from lot to lot? Could this be related to purity, presence of unlabeled phalloidin, or am I on the wrong track here?

- Should we test several lots in parallel with a standardized labeling assay (but this would be really expensive given the 300U smallest sold vial)? 

- Once we find a suitable lot, is there an alternative way to reconstitute/store the phalloidin to ensure preservation over months? I was thinking drying-down smaller aliquots with a suitable solvent and speedvac, like we do for fluorophores NHS-esters, but I'm not sure what solvent to use.

 

I know there are people from Molecular Probes/Thermo Fisher on this list, so any input from them would be also very helpful. I've also noticed that phalloidin-AF647 is available from another supplier (Cytoskeleton Inc.), not sure if these have the same production source, or if people have seen differences between suppliers.

 

Thanks,

 

Christophe

 

**********************************************************
Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues