Ca measurement in solution

[Balakrishnan KANNAN (IMCB)]

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Dear All,

This is not directly related to microscopy. I still believe that someone might be able to help.

We vary the amount of Ca in our protein solutions by having a constant amount of EGTA and add excess Ca to arrive at the level of Ca we require for our biochemical assays and TITF microscopy. To calculate the free Ca in this EGTA-buffered system we use Maxchealater (http://www.stanford.edu/~cpatton/webmaxc/webmaxclite115.htm).

With some extra bit of money made available to us, we are planning to buy a Ca-meter along with a Ca-electrode and a reference electrode. This will enable us to rule out the uncertainty in the Ca-levels arrived at by using the Maxchealater. When I explored I found Nova analytics (no commercial interest) have a Ca-meter and accessories. Unfortunately, I am not getting good pre-sales technical update from the local vendor.

I want to measure Ca-levels as low as 100 to 10 nanoMolar. 1 mM will be the upper end. Could someone help in identifying the appropriate Ca-meter?

Regards,
Bala

##################################################
Balakrishnan Kannan , Ph.D.,
Institute of Molecular and Cell Biology,
BR Lab, Proteos,
61, Biopolis Drive,
Singapore 138 673
e-mail: [hidden email]<mailto:[hidden email]>
Ph: [phone]
Fax: [phone]
##################################################



Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

[George McNamara]

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Hi Bala,

I cannot help on the electrode purchase, but have suggestions.

1. Molecular Probes (Invitrogen/Life Tech) has a 10 (11?) solution Ca++
calibration kit that is the "standard of care". Compare it to your home
made solutions.
2. Fura-2 (or modest loading of Fura-2/AM) is the live cell standard of
care. If you have a 340/380 nm ratio rig available, include it.
3. Take Nagai just published a review (in a Symposium issue on Calcium,
so issue may have additional articles of use):

Pérez Koldenkova V, Nagai T. Genetically encoded Ca(2+) indicators:
Properties and evaluation. Biochim Biophys Acta. 2013 Jan 22. doi:pii:
S0167-4889(13)00024-4. 10.1016/j.bbamcr.2013.01.011. [Epub ahead of
print] PubMed PMID: 23352808.

See in particular the Nano-CaMeleon series for very high affinity.

Note: Mg++ ions often compete with Ca++ - see the paper for more
information.


4. Extending range of Ca++ ion measurements in cells (and multiplexing
lots of things):

I figured out how to do multiplex fluorescent (and bioluminescent, so
Take's Nano-Lanterns are also "in play"). See Feb 5 (2013) ppt and pdf
inside the download at

http://works.bepress.com/gmcnamara/26/

The 4-curve graph (Okumoto ... Frommer 2005 PNAS) is on glutamate (which
is both a chinese food additive, neurotransmitter and the entry point of
glutamine into the Kreb/TCA cycle) but same deal applies to any affinity
series - including Calcium ions. A table lists 3 Ca++ ion sensors.

I am unaware of a Mg++ biosensor series, but my Tattletales concept
could by used with a wavelengths compatible conventional dye(s) to Mg++,
such as Mg-Fura-2. Spatial multiplexing in the pdf (abstract) and ppt
(poster) features loci in the cell nucleus, but tdPP7, tdMS2, PUF RNA
binding proteins (leading to PUFFRs: PUF fluorescent reporters, will
update the ppt in a few days with this), and simply targeting to
organelles (Rob Singer, peroxisomes, did this years ago in an early
MS2-GFP paper), can be used to make bright foci of reporters in the
cytoplasm.

I have placed Tattletales in the public domain (October 2012) and expect
to "reduce to practice" this summer after I move to a research lab at
MDACC (Houston) that has the TALEN/Tattletales robot that inspired the
idea. Speaking of positions - UMiami H.R. will (hopefully) post the
image core manager (director if anyone negotiates a new job title) on
its web site this week. I manage three confocal microscopes (LSM710,
Leica SP5, Leica MP/SP5/FCS/FLIM), a bunch of other microscopes, and
have a couple hundred users (not all simultaneously).

Sincerely,


George


George McNamara, Ph.D.
University of Miami, Miller School of Medicine (for now)
Cooper and Lee Immunotherapy lab, UT MDACC, Houston, TX (hopefully start
April 2013).




On 2/17/2013 9:29 PM, Balakrishnan KANNAN (IMCB) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> This is not directly related to microscopy. I still believe that someone might be able to help.
>
> We vary the amount of Ca in our protein solutions by having a constant amount of EGTA and add excess Ca to arrive at the level of Ca we require for our biochemical assays and TITF microscopy. To calculate the free Ca in this EGTA-buffered system we use Maxchealater (http://www.stanford.edu/~cpatton/webmaxc/webmaxclite115.htm).
>
> With some extra bit of money made available to us, we are planning to buy a Ca-meter along with a Ca-electrode and a reference electrode. This will enable us to rule out the uncertainty in the Ca-levels arrived at by using the Maxchealater. When I explored I found Nova analytics (no commercial interest) have a Ca-meter and accessories. Unfortunately, I am not getting good pre-sales technical update from the local vendor.
>
> I want to measure Ca-levels as low as 100 to 10 nanoMolar. 1 mM will be the upper end. Could someone help in identifying the appropriate Ca-meter?
>
> Regards,
> Bala
>
> ##################################################
> Balakrishnan Kannan , Ph.D.,
> Institute of Molecular and Cell Biology,
> BR Lab, Proteos,
> 61, Biopolis Drive,
> Singapore 138 673
> e-mail: [hidden email]<mailto:[hidden email]>
> Ph: [phone]
> Fax: [phone]
> ##################################################
>
>
>
> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.
>
>    

[Balakrishnan KANNAN (IMCB)]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi George,

Thanks for the suggestions. I am doing in vitro actin polymerization dynamics and use biochemical assays and TIRF microscopy to image the protein-protein interactions. Ca is one of the variables in the assay. I pre-mix Ca to the protein solutions and go on to do the assays. [Ca] is pre-decided and I only want to ensure that the [Ca] in the solution is what I wanted it to be. So I am exploring for a Ca-meter with appropriate electrode.

Nevertheless, your suggestions go a long way in planning my imaging in live cells in the future.

Best regards,
Bala

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Monday, February 18, 2013 7:44 PM
To: [hidden email]
Subject: Re: Ca measurement in solution ... Tattletales to multiplex many biosensors 9optionally affinity series) in live cells

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Bala,

I cannot help on the electrode purchase, but have suggestions.

1. Molecular Probes (Invitrogen/Life Tech) has a 10 (11?) solution Ca++
calibration kit that is the "standard of care". Compare it to your home
made solutions.
2. Fura-2 (or modest loading of Fura-2/AM) is the live cell standard of
care. If you have a 340/380 nm ratio rig available, include it.
3. Take Nagai just published a review (in a Symposium issue on Calcium,
so issue may have additional articles of use):

Pérez Koldenkova V, Nagai T. Genetically encoded Ca(2+) indicators:
Properties and evaluation. Biochim Biophys Acta. 2013 Jan 22. doi:pii:
S0167-4889(13)00024-4. 10.1016/j.bbamcr.2013.01.011. [Epub ahead of
print] PubMed PMID: 23352808.

See in particular the Nano-CaMeleon series for very high affinity.

Note: Mg++ ions often compete with Ca++ - see the paper for more
information.


4. Extending range of Ca++ ion measurements in cells (and multiplexing
lots of things):

I figured out how to do multiplex fluorescent (and bioluminescent, so
Take's Nano-Lanterns are also "in play"). See Feb 5 (2013) ppt and pdf
inside the download at

http://works.bepress.com/gmcnamara/26/

The 4-curve graph (Okumoto ... Frommer 2005 PNAS) is on glutamate (which
is both a chinese food additive, neurotransmitter and the entry point of
glutamine into the Kreb/TCA cycle) but same deal applies to any affinity
series - including Calcium ions. A table lists 3 Ca++ ion sensors.

I am unaware of a Mg++ biosensor series, but my Tattletales concept
could by used with a wavelengths compatible conventional dye(s) to Mg++,
such as Mg-Fura-2. Spatial multiplexing in the pdf (abstract) and ppt
(poster) features loci in the cell nucleus, but tdPP7, tdMS2, PUF RNA
binding proteins (leading to PUFFRs: PUF fluorescent reporters, will
update the ppt in a few days with this), and simply targeting to
organelles (Rob Singer, peroxisomes, did this years ago in an early
MS2-GFP paper), can be used to make bright foci of reporters in the
cytoplasm.

I have placed Tattletales in the public domain (October 2012) and expect
to "reduce to practice" this summer after I move to a research lab at
MDACC (Houston) that has the TALEN/Tattletales robot that inspired the
idea. Speaking of positions - UMiami H.R. will (hopefully) post the
image core manager (director if anyone negotiates a new job title) on
its web site this week. I manage three confocal microscopes (LSM710,
Leica SP5, Leica MP/SP5/FCS/FLIM), a bunch of other microscopes, and
have a couple hundred users (not all simultaneously).

Sincerely,


George


George McNamara, Ph.D.
University of Miami, Miller School of Medicine (for now)
Cooper and Lee Immunotherapy lab, UT MDACC, Houston, TX (hopefully start
April 2013).




On 2/17/2013 9:29 PM, Balakrishnan KANNAN (IMCB) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> This is not directly related to microscopy. I still believe that someone might be able to help.
>
> We vary the amount of Ca in our protein solutions by having a constant amount of EGTA and add excess Ca to arrive at the level of Ca we require for our biochemical assays and TITF microscopy. To calculate the free Ca in this EGTA-buffered system we use Maxchealater (http://www.stanford.edu/~cpatton/webmaxc/webmaxclite115.htm).
>
> With some extra bit of money made available to us, we are planning to buy a Ca-meter along with a Ca-electrode and a reference electrode. This will enable us to rule out the uncertainty in the Ca-levels arrived at by using the Maxchealater. When I explored I found Nova analytics (no commercial interest) have a Ca-meter and accessories. Unfortunately, I am not getting good pre-sales technical update from the local vendor.
>
> I want to measure Ca-levels as low as 100 to 10 nanoMolar. 1 mM will be the upper end. Could someone help in identifying the appropriate Ca-meter?
>
> Regards,
> Bala
>
> ##################################################
> Balakrishnan Kannan , Ph.D.,
> Institute of Molecular and Cell Biology,
> BR Lab, Proteos,
> 61, Biopolis Drive,
> Singapore 138 673
> e-mail: [hidden email]<mailto:[hidden email]>
> Ph: [phone]
> Fax: [phone]
> ##################################################
>
>
>
> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.
>
>

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.