Methods Digest, Vol 60, Issue 3

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Thu May 6 22:52:18 EST 2010


Question----
Answer,
Go through genetic engineering of Cry1Ac gene structure which has two
domains, the toxin- catalytic domain and second domain for
crystalization of the encoded protein. It may provide you some
important leads

On Thu, May 6, 2010 at 10:39 PM,  <methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. Western Blotting (Yvonne Couch)
>   2. question (gholamreza ahmadian)
>   3. Re: question (WS)
>   4. Re: Western Blotting (WS)
>   5. Re: Western Blotting (Dr Engelbert Buxbaum)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 5 May 2010 18:21:09 +0100
> From: "Yvonne Couch" <yvonne.couch from pharm.ox.ac.uk>
> Subject: Western Blotting
> To: <methods from magpie.bio.indiana.edu>
> Message-ID: <01b801caec77$5aaed0f0$100c72d0$@couch from pharm.ox.ac.uk>
> Content-Type: text/plain;       charset="us-ascii"
>
> Hi all,
>
>  Quick Western blotting question.in the past my Westerns have all been 'semi
>
>    quantitative' where I use an image programme like ImageJ to calculate
> the
>
>    intensity of the different bands.  Apparently it's possible to generate
>
>    standard curves for Western blots, does anyone have any experience of
> this
>
>    and can give me some general pointers?
>
>    Thanks in advance
>
>    Yvonne
>
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Wed, 5 May 2010 11:07:50 -0700 (PDT)
> From: gholamreza ahmadian <gholamrezaahmadian from yahoo.ca>
> Subject: question
> To: methods from magpie.bio.indiana.edu
> Message-ID: <988426.92354.qm from web31911.mail.mud.yahoo.com>
> Content-Type: text/plain; charset=us-ascii
>
> Dear All
> I made a mistake in saying it has 3 subunit.
> in fact it has 3 domain is correct. and it is one gene
> Thanks
> Reza
>
>
>
> ------------------------------
>
> Message: 3
> Date: Wed, 5 May 2010 12:17:38 -0700 (PDT)
> From: WS <novalidaddress from nurfuerspam.de>
> Subject: Re: question
> To: methods from net.bio.net
> Message-ID:
>        <d94797c5-6f80-414b-9adc-bac5bee817e0 from h9g2000yqm.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi Reza,
>
> trimerization for sure will increase the enzyme's activity. It is hard
> to say if the catalytic domain alone will show any activity. This will
> a lot depend on where you separate the domains and of course all
> domains influence each other. I would not suggest just to place the
> cut somewhere and hope that (at least some) activity might remain.
>
> If you are lucky, someone already has done this fragment analysis and
> published the results. If not, and you need this information, it will
> be up to you to perform these experiments.
>
> hope that helps
>
> Wo.
>
>
> ------------------------------
>
> Message: 4
> Date: Wed, 5 May 2010 12:29:37 -0700 (PDT)
> From: WS <novalidaddress from nurfuerspam.de>
> Subject: Re: Western Blotting
> To: methods from net.bio.net
> Message-ID:
>        <8e6358c9-ff79-4b78-96dd-4781b6cc61dc from j33g2000yqn.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Dear Yvonne,
>
> basically, you need to run standards in different amounts (like serial
> dilutions) and build a calibration curve. This needs to be done for
> every protein of your interest. Relative calibration, i.e. dilutions
> of a reference sample, eg a cell lysate, can do the job.
>
> ImageJ has calibration functions (thank's to Wayne Rasband's generous
> support). If you work with X-Ray films, the calibration will become
> very tedious, because the dynamic range is less than 2 decades and
> it's hard to expose the film in a way that all bands of interest are
> in the slope-range of the standard(s). Thus, I hope you have access to
> a more sophisticated instrument like an infrared fluorescence scanner
> with a higher dynamic range. Still a lot of manual work with ImageJ,
> but you will obtain data you may do statistics with.
>
> This topic also appears in the ImageJ mailing list from time to time,
> so you might find some more information there at
> https://list.nih.gov/cgi-bin/wa.exe?A0=IMAGEJ when you search for
> "western".
>
> Reagrds,
>
> Wo
>
>
>
> ------------------------------
>
> Message: 5
> Date: Thu, 6 May 2010 11:13:18 -0400
> From: Dr Engelbert Buxbaum <engelbert_buxbaum from hotmail.com>
> Subject: Re: Western Blotting
> To: methods from net.bio.net
> Message-ID: <MPG.264cb4a8e9180e23989685 from News.Individual.DE>
> Content-Type: text/plain; charset="us-ascii"
>
> In article <mailman.615.1273084973.25217.methods from net.bio.net>,
> yvonne.couch from pharm.ox.ac.uk says...
>>
>> Hi all,
>>
>>  Quick Western blotting question.in the past my Westerns have all been 'semi
>>  quantitative' where I use an image programme like ImageJ to calculate
>> the intensity of the different bands.  Apparently it's possible to
> generate
>>
>> standard curves for Western blots, does anyone have any experience of
>> this and can give me some general pointers?
>
> You will probably want to read the following
>
> @ARTICLE{Pit-07,
>  author       = {A. Pitre and Y. Pan and S. Pruett and O. Skalli},
>  title        = {On the use of ratio standard curves to accurately
> quantitate relative changes in protein levels by western blot},
>  journal      = {Anal. Biochem.},
>  volume       = {361},
>  year         = {2007},
>  pages        = {305-307},
>  doi          = {10.1016/j.ab.2006.11.008},
>  language     = {engl},
> }
>
>
>
> ------------------------------
>
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> End of Methods Digest, Vol 60, Issue 3
> **************************************
>



-- 
Dr V K Gupta
Sr Microbiologist (Mol Biology)
IMBL, Department of Entomology
Pun. Agric. Univ., Ludhiana (Pb)-141004- India
M: 081465-55515



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