slowing down or immobilizing (somewhat) purified fluorescent protein

[Jen Jackson]

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

Hello all,


I'm looking for a method to 'slow down', but not completely immobilize- a
low concentration of YFP particles to help characterize a TIRF system.
Ideally, we could perhaps later use this "YFP gel" to calibrate fluorescence
intensity of single YFP molecules in biological membranes, but for now I'm
looking for a decent way to have some sort of reduction in particle speed,
as compared to solution, without compromising the spectral properties (etc.)
of the protein.  Any tips, tricks?


Many thanks,

Jen

[Michelle Peckham]

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

Attach the YFP to the glass surface, using an antibody

On 28/06/2013 20:08, "Jen Jackson" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello all,
>
>
>I'm looking for a method to 'slow down', but not completely immobilize- a
>low concentration of YFP particles to help characterize a TIRF system.
>Ideally, we could perhaps later use this "YFP gel" to calibrate
>fluorescence
>intensity of single YFP molecules in biological membranes, but for now I'm
>looking for a decent way to have some sort of reduction in particle speed,
>as compared to solution, without compromising the spectral properties
>(etc.)
>of the protein.  Any tips, tricks?
>
>
>Many thanks,
>
>Jen

[Jen Jackson]

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

Thanks for your response.  I would prefer the molecules for the have even
more mobility so to test temporal resolution.  But antibodies are a great
start- can you recommend any protocol for attaching antibodies to a
coverslip (just use poly-d-lysine, or?).

[Michelle Peckham]

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

Hi again

This is taken from our Methods paper back in 2003 (Methods 29 (2003)
142­152)


Control coverslips, which were sparsely decorated
with single GFP fluorophores, were created by incubating
the coverslip with a low concentration of anti-
GFP antibody, blocking with 1 mg/ml bovine serum
albumin, and then incubating with a solution of about
10nM GFP. Coverslips were then viewed using TIRFM.
Individual GFP fluorophores could be identified as
separate objects that exhibited either one- or two-step
photobleaching (Fig. 7). These control slides were important
in setting up the TIRFM apparatus and in establishing
single-fluorophore sensitivity.



Hope that helps. If you want the whole paper - let me know.

Al best

Michelle

On 28/06/2013 21:06, "Jen Jackson" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Thanks for your response.  I would prefer the molecules for the have even
>more mobility so to test temporal resolution.  But antibodies are a great
>start- can you recommend any protocol for attaching antibodies to a
>coverslip (just use poly-d-lysine, or?).  

[Smith, Benjamin E.]

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

You may also be able to use a sieving matrix such as polyacrylamide or
low melting point agarose to spatially constrain the molecules.  
Depending on the pore size they still should rotate freely, but their
lateral diffusion should be greatly slowed.

If you find something that works, I'd love to know, as I also have a PI
who needs to slow down the Brownian motion of quantum dots while keeping
them in an aqueous solution.

-Ben Smith

Benjamin E. Smith, Ph.D.
Samuel Roberts Noble Microscopy Laboratory
Research Scientist II
University of Oklahoma
Norman, OK 73019
E-mail: [hidden email]
Voice   405-325-4391
FAX  [phone]
http://www.microscopy.ou.edu/

On 6/28/2013 3:06 PM, Jen Jackson wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks for your response.  I would prefer the molecules for the have even
> more mobility so to test temporal resolution.  But antibodies are a great
> start- can you recommend any protocol for attaching antibodies to a
> coverslip (just use poly-d-lysine, or?).

[Alfred Bahnson]

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

Jen et al,

If methylcellulose hasn't been ruled out, I want to try to clarify a bit.
 There are many different molecular weight formulations of methyl
cellulose, so when describing methods and concentrations it is necessary to
specify the particular type or source of methyl cellulose.  This is what I
did not do when our company patented use of methyl cellulose for
suppressing "non-biological" motion while assaying T-cell motility and
chemotaxis (patent number 06821747); don't try to read this, it's
embarrassing.  But the properties of methyl cellulose may provide just what
you are looking for.  Unlike a gel that returns to liquid when disrupted,
methyl cellulose maintains its viscosity when pipetted. Viscosity can be
adjusted by dilution.  And motion is impeded particularly alongside of
stationary objects, e.g. along the wall or surface of a vessel.

I recommend starting with Sigma's product literature:
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma-Aldrich/Product_Information_Sheet/m0262pis.Par.0001.File.tmp/m0262pis.pdf,
for
lack of a better reference at this time.

Good luck.

Al Bahnson
Kairos Instruments, LLC
Pittsburgh PA.
412-735-9983



On Fri, Jun 28, 2013 at 8:08 PM, Ben Smith <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> You may also be able to use a sieving matrix such as polyacrylamide or low
> melting point agarose to spatially constrain the molecules.  Depending on
> the pore size they still should rotate freely, but their lateral diffusion
> should be greatly slowed.
>
> If you find something that works, I'd love to know, as I also have a PI
> who needs to slow down the Brownian motion of quantum dots while keeping
> them in an aqueous solution.
>
> -Ben Smith
>
> Benjamin E. Smith, Ph.D.
> Samuel Roberts Noble Microscopy Laboratory
> Research Scientist II
> University of Oklahoma
> Norman, OK 73019
> E-mail: [hidden email]
> Voice   405-325-4391
> FAX  [phone]
> http://www.microscopy.ou.edu/
>
>
> On 6/28/2013 3:06 PM, Jen Jackson wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Thanks for your response.  I would prefer the molecules for the have even
>> more mobility so to test temporal resolution.  But antibodies are a great
>> start- can you recommend any protocol for attaching antibodies to a
>> coverslip (just use poly-d-lysine, or?).
>>
>

[Steffen Dietzel]

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

Maybe I am missing something here, but how about just adding glycerol to
the solution? Other options might be a low concentration of
low-melting-point agarose or a gel used for ultrasonic examinations.

Steffen

On 28.06.2013 21:08, Jen Jackson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
>
>
> I'm looking for a method to 'slow down', but not completely immobilize- a
> low concentration of YFP particles to help characterize a TIRF system.
> Ideally, we could perhaps later use this "YFP gel" to calibrate fluorescence
> intensity of single YFP molecules in biological membranes, but for now I'm
> looking for a decent way to have some sort of reduction in particle speed,
> as compared to solution, without compromising the spectral properties (etc.)
> of the protein.  Any tips, tricks?
>
>
> Many thanks,
>
> Jen
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Jen Jackson]

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

Thanks all for your input!  

In regards to something like glycerol- for my purposes, the high refractive
index will cause problems with TIR.  So my question is now clearer: which
gels would act as the best imaging medium for TIRF?  Cygel was mentioned to
have a refractive index of 1.36, so could be a good candidate.  However it's
also somewhat expensive, so if anyone has used agarose, or other materials
suggested in this thread- successfully with TIRF microscopy- I would be
interested to hear about it.

Thanks!

Jen