Proteins Digest, Vol 29, Issue 1

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)


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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: [Only registered users see links. ]
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



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Message: 2
Date: Mon, 01 Oct 2007 16:17:00 GMT
From: Marvin

Subject: [Protein-analysis] Re: Drug binding: When fluorescence
polarization fails
To: [Only registered users see links. ]
Message-ID: <049Mi.11183$bV2.6674@trndny02>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Dominic-Luc Webb wrote:

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.


It could be that the change of solvent to 50% glycerol
reduces the fluorescence lifetime, thus increasing the
measured polarization.



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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: [Only registered users see links. ]
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





------------------------------

Message: 4
Date: Tue, 02 Oct 2007 11:21:58 +0200
From: Patrick Sticher
Subject: [Protein-analysis] Course on Biomolecular Modelling
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ]>
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
[Only registered users see links. ]

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
[Only registered users see links. ]

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
Mail [Only registered users see links. ]



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