[Protein-analysis] Re: Proteins Digest, Vol 29, Issue 1

Rhiannon Evans via proteins%40net.bio.net (by EvansRM8 from cf.ac.uk)
Thu Oct 4 03:35:45 EST 2007


Hi Sai,

we use the stratagene quick change kit in our lab, it works a treat  
providing you have the correct conditions for your PCR in the first  
place.
I often find the best way to figure this out is to do a temperature  
gradient, a Mg gradient and a DMSO gradient. Although there is MgSO4  
in the buffer i still needed to add an extra 2 mM to my samples. DMSO  
helps with preventing the formation of secondary structures also.

Good luck!

Rhiannon
************************************************
Rhiannon Mari Evans
RKA Group
School of Chemistry
Cardiff University
Main Building
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Cardiff
CF10 3AT

Mobile: 07828 156417
Office/Lab : 029 20 879417
Email: EvansRM8 from cf.ac.uk

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On 2 Oct 2007, at 21:39, Sai Praveen wrote:

> Hi,
>
>   I am planning to perform site-directed mutagenesis using PCR  
> megaprimer  (~60bp). I was quite attracted by the Stratagene Quik- 
> Change site-directed mutagenesis kit's simplicity. Please, if you  
> had a chance to work with this kit, let me know if you had trouble  
> with primer forming secondary structures.
>
>   Thanks!
>   Sai
>
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> Today's Topics:
>
> 1. Drug binding: When fluorescence polarization fails
> (Dominic-Luc Webb)
> 2. Re: Drug binding: When fluorescence polarization fails (Marvin)
> 3. Re: Drug binding: When fluorescence polarization fails
> (Dominic-Luc Webb)
> 4. Course on Biomolecular Modelling (Patrick Sticher)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 1 Oct 2007 16:05:01 +0200
> From: Dominic-Luc Webb
> Subject: [Protein-analysis] Drug binding: When fluorescence
> polarization fails
> To: proteins from net.bio.net
> Message-ID:
>
> Content-Type: TEXT/PLAIN; charset=US-ASCII
>
>
> I have worked for some time with fluroescence polarization (FP)
> in studies of drug-protein binding. My drug, which is
> hydrophobic, typically has a fluorescent adduct and the
> solution has 0.01% Tween 20 to keep down non-specific binding
> to test tubes, multiwell plates, etc. Assay usually works well.
>
> I usually do a check with a series of glycerol concentrations
> to ensure that FP is working OK. I expect at high glycerol
> concentration, the higher viscosity lowers molecular rotation
> which is detected as an increase in the amount of polarized
> light in the static (parallel) channel. This usually works
> fine, fluorescein for instance. I recently have found some
> labelled drugs that show no increase in polarization, and
> even a small decrease (about 20 mP) even when adding glycerol
> to 50%. Again, Tween 20 is 0.01% throughout. Fluorescence
> intensity remains relatively constant with only a slight
> parallel increase in both channels. The underlying physics of
> FP always seemed pretty straightforward, but now I am
> confronted with such data that has been reproduced several
> times and it is not clear how this came about.
>
> Strangely, when a protein that is known to be a drug target is
> present, there is a strong increase in polarization (100-300 mP).
> The glycerol data has created an uncertainty about negative
> data for test proteins that I am screening for drug binding.
>
> Is there anyone in here that might have some hints how this
> lack of change in polarization in glycerol might have come
> about, maybe some suggestions how to troubleshoot this?
>
> Dominic
>
>
>
> ------------------------------
>
> Message: 2
> Date: Mon, 01 Oct 2007 16:17:00 GMT
> From: Marvin
>
> Subject: [Protein-analysis] Re: Drug binding: When fluorescence
> polarization fails
> To: proteins from net.bio.net
> Message-ID: <049Mi.11183$bV2.6674 from trndny02>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Dominic-Luc Webb wrote:
>> I have worked for some time with fluroescence polarization (FP)
>> in studies of drug-protein binding. My drug, which is
>> hydrophobic, typically has a fluorescent adduct and the
>> solution has 0.01% Tween 20 to keep down non-specific binding
>> to test tubes, multiwell plates, etc. Assay usually works well.
>>
>> I usually do a check with a series of glycerol concentrations
>> to ensure that FP is working OK. I expect at high glycerol
>> concentration, the higher viscosity lowers molecular rotation
>> which is detected as an increase in the amount of polarized
>> light in the static (parallel) channel. This usually works
>> fine, fluorescein for instance. I recently have found some
>> labelled drugs that show no increase in polarization, and
>> even a small decrease (about 20 mP) even when adding glycerol
>> to 50%. Again, Tween 20 is 0.01% throughout. Fluorescence
>> intensity remains relatively constant with only a slight
>> parallel increase in both channels. The underlying physics of
>> FP always seemed pretty straightforward, but now I am
>> confronted with such data that has been reproduced several
>> times and it is not clear how this came about.
>>
>> Strangely, when a protein that is known to be a drug target is
>> present, there is a strong increase in polarization (100-300 mP).
>
> That is what I'd expect. Binding to a large molecule (i.e.,
> the protein) reduces the rotation rate of the labeled drug,
> thus increasing the polarization.
>
>> The glycerol data has created an uncertainty about negative
>> data for test proteins that I am screening for drug binding.
>>
>> Is there anyone in here that might have some hints how this
>> lack of change in polarization in glycerol might have come
>> about, maybe some suggestions how to troubleshoot this?
>
> It could be that the change of solvent to 50% glycerol
> reduces the fluorescence lifetime, thus increasing the
> measured polarization.
>
>>
>> Dominic
>>
>
>
> ------------------------------
>
> Message: 3
> Date: Tue, 2 Oct 2007 11:11:06 +0200
> From: Dominic-Luc Webb
> Subject: [Protein-analysis] Re: Drug binding: When fluorescence
> polarization fails
> To: proteins from net.bio.net
> Message-ID:
>
> Content-Type: TEXT/PLAIN; charset=US-ASCII
>
>
> Thanks Marvin for your response, and sorry about any confusion I
> might have created in the use of the word "strangely". The
> "strangeness" had only to do with the disjunct between the
> unexpected glycerol data and the expected protein binding data
> using the same labelled drug. Until now, I regarded both of
> these as positive control tests.
>
> Regarding 50% glycerol, I could be convinced that the fluorescence
> lifetime was changed, but I would need to bring this in line with
> recent experiments. I went back and made some further measurements
> and quantifications together with data I collected earlier. I
> calculate a 1.4 fold increase in fluorescence intensity (FI) when
> measured with normal FI filter set. Using the FP filters to measure
> same samples, there was also an increase in fluorescence intensity in
> both of the two channels (1.35 in the static channel, 1.41 in the
> perpendicular channel). Fluorescence yield must have increased
> concommitantly with a differential shift towards the perpendicular
> channel.
>
> Considering that last sentence, this assay has thus far not been
> very sensitive to different intensities (e.g., fluorescein over a
> few orders of magnitude). However, the differential shift towards
> the perpendicular channel (1.41/1.35), although small, approaches
> significance and seems in line with your suggestion that fluorescence
> lifetime was decreased. Does this make sense?
>
> Dominic
>
>
>>> Strangely, when a protein that is known to be a drug target is
>>> present, there is a strong increase in polarization (100-300 mP).
>>
>> That is what I'd expect. Binding to a large molecule (i.e.,
>> the protein) reduces the rotation rate of the labeled drug,
>> thus increasing the polarization.
>>
>>> The glycerol data has created an uncertainty about negative
>>> data for test proteins that I am screening for drug binding.
>>>
>>> Is there anyone in here that might have some hints how this
>>> lack of change in polarization in glycerol might have come
>>> about, maybe some suggestions how to troubleshoot this?
>>
>> It could be that the change of solvent to 50% glycerol
>> reduces the fluorescence lifetime, thus increasing the
>> measured polarization.
>>
>>>
>>> Dominic
>>>
>>
>
>
>
> ------------------------------
>
> Message: 4
> Date: Tue, 02 Oct 2007 11:21:58 +0200
> From: Patrick Sticher
> Subject: [Protein-analysis] Course on Biomolecular Modelling
> To: proteins from magpie.bio.indiana.edu
> Message-ID: <47020DB6.5020504 from bioc.unizh.ch>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Dear colleagues,
>
> please be informed that applications for the
>
> 6th NCCR PRACTICAL COURSE ON BIOMOLECULAR MODELLING
>
> will be accepted still until October 10, 2007.
>
> The course will take place January 6 - 11, 2008 as a winter retreat in
> the Swiss Alps, and will cover key topics in the area of computational
> structural biology. The course format includes morning lectures by
> experts in the field that alternate with later afternoon to evening
> tutorials and discussions.
>
> For further details about this course, please visit the course website
> http://www.structuralbiology.unizh.ch/course2008.asp
>
> If you are interested in this course, please use the online form on  
> the
> course website to apply.
>
> Best regards,
> Patrick Sticher
>
> -- 
> _________________________________
> Visit the NCCR on the Internet
> www.structuralbiology.uzh.ch
>
> Dr. Patrick Sticher Moser
> NCCR Scientific Officer
> Institute of Biochemistry
> University of Zürich
> Winterthurerstrasse 190
> CH - 8057 Zürich
>
> Phone +41 / (0)44 / 635 54 84
> Fax +41 / (0)44 / 635 59 08
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>
>
>
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> End of Proteins Digest, Vol 29, Issue 1
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