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, <[email protected]> wrote: > Send Methods mailing list submissions to > [email protected] > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > [email protected] > > You can reach the person managing the list at > [email protected] > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > 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" <[email protected]> > Subject: Western Blotting > To: <[email protected]> > Message-ID: <01b801caec77$5aaed0f0$100c72...@[email protected]> > 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 <[email protected]> > Subject: question > To: [email protected] > Message-ID: <[email protected]> > 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 <[email protected]> > Subject: Re: question > To: [email protected] > Message-ID: > <[email protected]> > 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 <[email protected]> > Subject: Re: Western Blotting > To: [email protected] > Message-ID: > <8e6358c9-ff79-4b78-96dd-4781b6cc6...@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 <[email protected]> > Subject: Re: Western Blotting > To: [email protected] > Message-ID: <[email protected]> > Content-Type: text/plain; charset="us-ascii" > > In article <[email protected]>, > [email protected] 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}, > } > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 60, Issue 3 > ************************************** > -- Dr V K Gupta Sr Microbiologist (Mol Biology) IMBL, Department of Entomology Pun. Agric. 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