[FDS] Re: FDS sensitivity

[Tom Laue]

Hi Steve-
The only change to the manual would be that (for the time being) one has 
to leave the "Focus and alignment" form after performing the radial 
calibration, then re-enter it to perform the focus calibration. Once the 
vertical calibration is set, you may want to check it periodically 
(every few months or if the sensitivity takes a dive), but it should 
remain constant.
Fluorescein will photo-bleach over time. The amount of exposure to light 
in the FDS is fairly small, but if the centerpiece has been kept in room 
light, it may have bleached some. It is a pain to fill correctly (high 
on our to do list). I wouldn't recommend changing it unless it fails to 
give a magnet angle.
Best wishes,
Tom

smcbryan wrote:
> Tom, is the radial and focus calibration procedure accurately described in the 
> AOS manual? Or is there a document describing this I can access? Can this 
> become a regular part of instrument upkeep, ie performing this procedure 
> monthly?
> Obviously, we would all like to get the most sensitivity our of our machines. 
> My only real check of signal/sensitivity is the calibration centerpiece I 
> loaded with free fluorosceinin january, and I wonder if this may not 
> accurately represent the anticipated signal strength anymore. 
> thanks, 
> steve
>
>
>   
>> ===== Original Message From Tom.Laue at unh.edu, AUC Fluorescence Detection 
>>     
> System List <fds at biochem.uthscsa.edu> =====
>   
>> Hi all-
>> At 600 nM, the Alexa488 IgG should blast the detector, and very, very
>> low gains should be required. We have gotten usable data at 1 pM with
>> Alex488 IgG.
>> Thanks to VPN, I was able to watch your instrument gathering data. It
>> seems to be working fine, but the sensitivity absolutely stinks. My
>> guess is that the objective lens is not focused properly. A small amount
>> of mis-focus can have a big effect on sensitivity. Virgil went through
>> the radial and focus calibration procedure correctly, but I think there
>> may be a bug in the program that requires that these two calibrations be
>> done separately. That is, you should do the radial calibration first,
>> then exit the Focus and calibration form. The focus calibration should
>> be done by re-opening the form. This "wake up and go to sleep" procedure
>> shouldn't be necessary, but for some reason is. It has been logged in
>> Bugzilla. Sorry!
>> Two other tips. First, the samples should be degassed before running. We
>> have seen the fluorescence increase by an order of magnitude following
>> degassing (this is not what is affecting your experiments). The second
>> is to include a small amount of a carrier protein. We have used 0.1
>> mg/ml ovalbumin, BSA or (recently) kappa casein as a carrier. Some
>> proteins do not require a carrier, but some- particularly IgG- will
>> plaster themselves on the windows, walls or at the meniscus. Again, at
>> 600 nM, the IgG acts like its own carrier protein- you should have had
>> light igniting the detector.
>> We have a small amount of GFP that we are using for some GFP/anti GFP
>> experiments in serum. If any of you are interested (and there is enough)
>> I will ask Kari to send you some in TBS + 0.1 mg/ml ovalbumin. I was
>> just looking at some data from a 40 nM sample (it has some aggregate).
>> The signal was 2500 and 2800 counts at a gain of 70% and PGA setting of
>> 2 for 2 different samples. These values will vary depending on: 1) the
>> PMT gain, 2) the PGA setting, 3) the strength of the laser, 4) the
>> sensitivity of the PMT, 5) the angle over which data are acquired from
>> the sample, 6) quenchers in the sample and 7) how much of the material
>> is lost to walls. So, for example, the data from the same sample at the
>> same gain settings, but in another cell, gave 1500 counts. These data
>> were acquired in serum, so it is hard to know what the cause of the
>> difference is.
>> Kari is about to do another run with this GFP. I've asked her to do a
>> run of just the 40 nM GFP in TBS+ 0.1 mg/ml ovalbumin. We'll post those
>> data so we can compare system sensitivities.
>> Best wishes,
>> Tom
>>
>> Borries Demeler wrote:
>>     
>>> Tom & Virgil,
>>>
>>> We are obviously still stumped on the intensity issue with our
>>> machine. A sample of Alexa 488 labeled sample that emits at
>>> 30,000 counts on a different spectrophotometer at 600 nM concentraiton
>>> is giving us barely 700 (very noisy!) counts at full gain settings.
>>>
>>> Dilutions of this protein reduce further in intensity. Even if carrier
>>> protein were added, 600 nm is so concentrated we should get more signal.
>>>
>>> It is my understanding that you will send us a GFP sample that we can
>>> use as a standard to compare to your machine. Could you post some
>>> velocity scans of THIS sample so we can see what we should be able to
>>> get?
>>>
>>> Is there something about Alexa 488 that doesn't make it show up
>>> well in the XLF?
>>>
>>> Thanks, -Borries
>>>
>>> _______________________________________________
>>> FDS mailing list
>>> FDS at biochem.uthscsa.edu
>>> http://biochem.uthscsa.edu/mailman/listinfo/fds
>>>
>>>
>>>       
>> --
>> Department of Biochemistry and Molecular Biology
>> University of New Hampshire
>> Durham, NH 03824-3544
>> Phone: 603-862-2459
>> FAX:   603-862-0031
>> E-mail: Tom.Laue at unh.edu
>> www.bitc.unh.edu
>> www.camis.unh.edu
>> _______________________________________________
>> FDS mailing list
>> FDS at biochem.uthscsa.edu
>> http://biochem.uthscsa.edu/mailman/listinfo/fds
>>     
>
> Steven J McBryant, PhD
> Director: Biomolecular Ultracentrifugation Facility
> Department of Biochemistry and Molecular Biology
> Colorado State University
> 970-491-5586
>
>   

-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu