Calculating protein concentration

Roman Mezencev via methods%40net.bio.net (by rmezencev3 from gatech.edu)
Wed Jan 23 13:55:00 EST 2008


In my view this discussion starts to be a bit counterproductive. It probably
belongs to a completely different forum. Also, blaming only teachers for that
may be an oversimplification. It is a student's responsibility to make sure
that s/he understands basic concepts before s/he applies to a
college/university. It should be a teacher's responsibility to make sure that a
student with such a lack of knowledge would not pass. I suggest that this forum
adhere to its skope which is techniques in cellular/molecular biology and
biochemistry.

RM




Quoting methods-request from oat.bio.indiana.edu:

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> Today's Topics:
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>    1. Re: Calculating Protein Concentration (Pow Joshi)
>    2. Re: Calculating Protein Concentration (DK)
>    3. Re: gateway system (DK)
>    4. Re: Calculating Protein Concentration (Sudheendra Rao N R)
>    5. Re: Gateway System (Felipe da Veiga Leprevost)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Tue, 22 Jan 2008 15:54:33 -0500
> From: "Pow Joshi" <pow.joshi from gmail.com>
> Subject: Re: Calculating Protein Concentration
> To: "Tom Anderson" <ucgatan from ucl.ac.uk>
> Cc: methods from magpie.bio.indiana.edu
> Message-ID:
> 	<710764ea0801221254j1cc14490p1405e080f11bdc96 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> On 22/01/2008, Tom Anderson <ucgatan from ucl.ac.uk> wrote:
> >
> > On Mon, 21 Jan 2008, Pow Joshi wrote:
> >
> > > On 21/01/2008, DK <dk from no.email.thankstospam.net> wrote:
> > >
> > > > That you apparently had bad teachers who allowed you to successfully
> > > > go through things that require this knowledge without acquiring it is
> > > > most certainly not your problem and I don't think any single reply
> > > > said that. It's a tragedy of many modern societies. And I am afraid we
> > > > will be paying dearly for it down the road. Dumbing down higher
> > > > education is one of the shortest ways to a societal suicide.
> > >
> > > ...and this is a general comment:  I one also sees very clearly, that
> > > science is , as again someone has already suggested, fast becoming a
> > > game of numbers and rote "doing" rather than anything to do with any
> > > kind of thought process or intellect that gives rise to hypothesis and
> > > predictions....  it is as well that one invented robots to do ones
> > > bidding (which may, very well, replace the numerous postdocs and
> > > technicians that the universities and industries employ) ... it is the
> > > age of "brute force" I suppose....
> >
> > I hear this a lot. The funny thing is, it doesn't correspond in the
> > slightest to the way i, a graduate student, do science, or the way i see
> > anyone else around me, student or postdoc, doing science. Yes, we use
> > kits, and i doubt many people could tell you exactly what buffer QC does,
> > but i can assure you we're fully engaged on the problems we work on. Not
> > having to think about how DNA binds to a column doesn't mean we do less
> > thinking, just that we have to spend more time thinking about the other
> > problems we face.
>
>
>
>
> So, what's going on here? Is it the case that science is descending into
> > rote doing or not? Is this just a standard complaint of grumpy old
> > scientists? Have i been lucky to work in more switched-on environments? Is
> > this something that's common in, say, biochemistry, or structural biology,
> > or molecular biology labs, but less so in cell biology, where i work?
> > Could it perhaps be a difference in culture between the US (where i think
> > a lot of you are, although i'm not sure) and the UK (where i am)?
>
>
>
>
>
> It would be grimly ironic if it was the latter. Group leaders here are
> > constantly urging us larvae to postdoc in the US, to learn the 'American
> > style of doing science', implying it's some master secret that will unlock
> > successful careers. Perhaps it's the exact opposite.
> >
> > I should say, though, that i don't think that's the case; British people i
> > know who've worked in US labs generally report that there is no
> > significant difference in culture (modulo the average size of labs, which
> > is much bigger in the US). I suspect it's a weird cargo cult that's taken
> > hold amongst British group leaders: all of them went to the US to postdoc,
> > and now they're successful, so of course if we do it, we will be too,
> > right?  This displays a woeful lack of logic and understanding of cause
> > and effect, but hey, this is group leaders we're talking about, no news
> > there.
>
>
>
> ah, Tom, the silly little details of knowiing "how the DNA binds to a
> column" actually helps one understand and interpret the results , and
> ofcourse, troubleshoot ....and believe me, it is what constitutes "thinking"
> as well, not just the the wonderful wild trips of imagination which we all
> love to indulge in .........and no, I don't consider myself grumpy little
> scientist, please, my grad-schooling was not long ago for such luxuries
> ......and the again some of your and my british colleagues may beg to differ
> from your view of the wonderful side of the coin....
> .... my comments were a homage to all the lovely literature that gets
> published, a lot of which is so unique that not even the good Mother  Nature
> will ever reproduce it ....
>
>
>
>
> tom
> >
> > --
> > Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E
> > 6BT
> > (t) +44 (20) 76797264   (f) +44 (20) 76797805   (e)
> > thomas.anderson from ucl.ac.uk
> >
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
> ------------------------------
>
> Message: 2
> Date: Wed, 23 Jan 2008 00:58:00 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: Calculating Protein Concentration
> To: methods from net.bio.net
> Message-ID: <qiwlj.721$QH2.438 from newsfe05.lga>
>
> In article <mailman.264.1201029616.2451.methods from net.bio.net>, Tom Anderson
> <ucgatan from ucl.ac.uk> wrote:
> >On Mon, 21 Jan 2008, Pow Joshi wrote:
> >
> >> On 21/01/2008, DK <dk from no.email.thankstospam.net> wrote:
> >>
> >> > That you apparently had bad teachers who allowed you to successfully
> >> > go through things that require this knowledge without acquiring it is
> >> > most certainly not your problem and I don't think any single reply
> >> > said that. It's a tragedy of many modern societies. And I am afraid we
> >> > will be paying dearly for it down the road. Dumbing down higher
> >> > education is one of the shortest ways to a societal suicide.
> >>
> >> ...and this is a general comment:  I one also sees very clearly, that
> >> science is , as again someone has already suggested, fast becoming a
> >> game of numbers and rote "doing" rather than anything to do with any
> >> kind of thought process or intellect that gives rise to hypothesis and
> >> predictions....  it is as well that one invented robots to do ones
> >> bidding (which may, very well, replace the numerous postdocs and
> >> technicians that the universities and industries employ) ... it is the
> >> age of "brute force" I suppose....
> >
> >I hear this a lot. The funny thing is, it doesn't correspond in the
> >slightest to the way i, a graduate student, do science, or the way i see
> >anyone else around me, student or postdoc, doing science.
>
> When I came to the lab, everyone was religiously was dissolving
> agarose in QG/QC at 55C, wasting precious time of the single
> water bath. No one bothered to check what happens if one
> 1) tries 37C, 2) tries 68C (for which there is a heating block
> available). The answer - nothing happens, works exactly the
> same way as 55C. Slower at 37, faster at 68.
>
> Further, even in the light of indisputable evidence that "Quik
> solution" is DMSO, many were reluctant to use DMSO and
> kept buying pointless because they could not buy "Quik
> solution".
>
> Try asking why we have a stirring bar in dialysis or why
> it is customary to flame flasks' and bottles' necks when
> working with sterile cultures. Betcha few in your enlightened
> environment know the answers - they just treat this stuff as
> black boxes and protocols. (Resulting in absolutely ridiculous
> behavior when, for example, flaming things).
>
> >Yes, we use
> >kits, and i doubt many people could tell you exactly what buffer QC does,
>
> Guanidinium thiocyanate - a salt of two chaotropic ions.
> PB, guanidinium hydrochloride, is not soluble at concentrations
> needed to dissolve agarose. More chaotropic salt does it.
>
> >but i can assure you we're fully engaged on the problems we work on. Not
> >having to think about how DNA binds to a column doesn't mean we do less
> >thinking, just that we have to spend more time thinking about the other
> >problems we face.
>
> Yeah, except when one loses DNA and has no idea what happened.
> Or when one faces situation that cannot be circumvented by buying
> yet another kit.
>
> >So, what's going on here? Is it the case that science is descending into
> >rote doing or not? Is this just a standard complaint of grumpy old
> >scientists? Have i been lucky to work in more switched-on environments? Is
> >this something that's common in, say, biochemistry, or structural biology,
> >or molecular biology labs, but less so in cell biology, where i work?
>
> Cell biology, where I used to work, is absolutely the worst in this
> respect! (And most bullshit published, BTW; e.g. "colocalization"
> pics that show ~ entire cytoplasm stained with two colors; or
> "binding" and "interaction" based on IPs where totals are
> omitted to cover up the fact that barely anything is pulled down
> and tiny slices of the gels are shown to disguise horribly
> non-specific antibodies).
>
> >Could it perhaps be a difference in culture between the US (where i think
> >a lot of you are, although i'm not sure) and the UK (where i am)?
>
> Could be but don't forget where the original poster comes from.
>
> >It would be grimly ironic if it was the latter. Group leaders here are
> >constantly urging us larvae to postdoc in the US, to learn the 'American
> >style of doing science', implying it's some master secret that will unlock
> >successful careers. Perhaps it's the exact opposite.
> >
> >I should say, though, that i don't think that's the case; British people i
> >know who've worked in US labs generally report that there is no
> >significant difference in culture (modulo the average size of labs, which
> >is much bigger in the US).
>
> No difference in culture but an appreciable difference in education
> system. May still be a factor.
>
> > suspect it's a weird cargo cult that's taken
> >hold amongst British group leaders: all of them went to the US to postdoc,
> >and now they're successful, so of course if we do it, we will be too,
> >right?  This displays a woeful lack of logic and understanding of cause
> >and effect, but hey, this is group leaders we're talking about, no news
> >there.
>
> Now that the USA is, unfortunately, losing its status of world's
> scientific leader (I remember reading couple years ago in Science
> that European papers now have higher citation impact factor),
> this too will pass. French used to come to the USA for postdoc
> to boost their chances home; this is largely no longer the
> case today.
>
> DK
>
>
> ------------------------------
>
> Message: 3
> Date: Wed, 23 Jan 2008 01:10:12 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: gateway system
> To: methods from net.bio.net
> Message-ID: <Ttwlj.722$QH2.12 from newsfe05.lga>
>
> In article
> <3ba47416-21de-46a3-a220-31222b988734 from q39g2000hsf.googlegroups.com>, "|
> |Koen" <k.vd.wetering from nki.nl> wrote:
> >Dear all,
> >
> >I want to use the gateway system of invitrogen to minimize the
> >workload of subcloning our cDNAs in various expression vectors.
>
> Have you considered the fact that the cloning artifacts that it
> inevitably reproduces (please correct me if I am wrong) may
> skew your results significantly?
>
> I've seen more than one example of just a single "strange"
> amino acid significantly affecting protein's properties.
>
> Before
> >using the system I have to clone our cDNAs in an appropriate pEntr
> >vector. I used pEntr1a for this but I do not get any colonies on
> >kanamycin plates using homemade DH5alpha or Invitrogen Top10 cells. I
> >did remove the ccdB sequence before putting in our cDNA. I also tested
> >cutting out the ccdB sequence, religating the empty vector and
> >transforming bacteria with this. I get only a very limited amount of
> >colonies from transformed Top10 cells and none on our own competent
> >DH5alpha.
> >
> >can anyone tell me what I did wrong?
>
> So, if I understand correctly, you are not getting ligation for cloning
> into pEntry clone to work? Is this right? If yes, then, unfortunately,
> there is almost an endless list of possibilities and, unless you have
> done all the usual ligation troubleshooting controls and can list
> them, there is no way for others to know what went wrong.
>
> If you are talking about recombination from pEntry into
> other vectors not working then, since this would be using a kit for
> which very little concrete information is available, it's hard to tell
> where problems are. The simplest is that you have dirty miniprep
> that carries over some inhibitors into the reaction. Try cleaning
> up your vector.
>
> DK
>
>
> ------------------------------
>
> Message: 4
> Date: Wed, 23 Jan 2008 20:39:25 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Re: Calculating Protein Concentration
> To: "Pow Joshi" <pow.joshi from gmail.com>, methods
> 	<methods from magpie.bio.indiana.edu>
> Message-ID:
> 	<a1a1abbb0801230709l4254432dl4c3b466dc2320ad4 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> great going :)
> i guess we all need little patience..
> those who are wondering as to how did some people manage to pass need to
> accept that they have passed..
> and those who are wondering why people are responding this way..need to get
> little numb..
>
> regards.
> its a great forum
>
> Sudheendra.
>
> On Jan 23, 2008 2:24 AM, Pow Joshi <pow.joshi from gmail.com> wrote:
>
> > On 22/01/2008, Tom Anderson <ucgatan from ucl.ac.uk> wrote:
> > >
> >  > On Mon, 21 Jan 2008, Pow Joshi wrote:
> > >
> > > > On 21/01/2008, DK <dk from no.email.thankstospam.net> wrote:
> > > >
> > > > > That you apparently had bad teachers who allowed you to successfully
> > > > > go through things that require this knowledge without acquiring it
> > is
> > > > > most certainly not your problem and I don't think any single reply
> > > > > said that. It's a tragedy of many modern societies. And I am afraid
> > we
> > > > > will be paying dearly for it down the road. Dumbing down higher
> > > > > education is one of the shortest ways to a societal suicide.
> > > >
> > > > ...and this is a general comment:  I one also sees very clearly, that
> > > > science is , as again someone has already suggested, fast becoming a
> > > > game of numbers and rote "doing" rather than anything to do with any
> > > > kind of thought process or intellect that gives rise to hypothesis and
> > > > predictions....  it is as well that one invented robots to do ones
> > > > bidding (which may, very well, replace the numerous postdocs and
> > > > technicians that the universities and industries employ) ... it is the
> > > > age of "brute force" I suppose....
> > >
> > > I hear this a lot. The funny thing is, it doesn't correspond in the
> > > slightest to the way i, a graduate student, do science, or the way i see
> > > anyone else around me, student or postdoc, doing science. Yes, we use
> > > kits, and i doubt many people could tell you exactly what buffer QC
> > does,
> > > but i can assure you we're fully engaged on the problems we work on. Not
> > > having to think about how DNA binds to a column doesn't mean we do less
> > > thinking, just that we have to spend more time thinking about the other
> > > problems we face.
> >
> >
> >
> >
> > So, what's going on here? Is it the case that science is descending into
> > > rote doing or not? Is this just a standard complaint of grumpy old
> > > scientists? Have i been lucky to work in more switched-on environments?
> > Is
> > > this something that's common in, say, biochemistry, or structural
> > biology,
> > > or molecular biology labs, but less so in cell biology, where i work?
> > > Could it perhaps be a difference in culture between the US (where i
> > think
> > > a lot of you are, although i'm not sure) and the UK (where i am)?
> >
> >
> >
> >
> >
> > It would be grimly ironic if it was the latter. Group leaders here are
> > > constantly urging us larvae to postdoc in the US, to learn the 'American
> > > style of doing science', implying it's some master secret that will
> > unlock
> > > successful careers. Perhaps it's the exact opposite.
> > >
> > > I should say, though, that i don't think that's the case; British people
> > i
> > > know who've worked in US labs generally report that there is no
> > > significant difference in culture (modulo the average size of labs,
> > which
> > > is much bigger in the US). I suspect it's a weird cargo cult that's
> > taken
> > > hold amongst British group leaders: all of them went to the US to
> > postdoc,
> > > and now they're successful, so of course if we do it, we will be too,
> > > right?  This displays a woeful lack of logic and understanding of cause
> > > and effect, but hey, this is group leaders we're talking about, no news
> > > there.
> >
> >
> >
> > ah, Tom, the silly little details of knowiing "how the DNA binds to a
> > column" actually helps one understand and interpret the results , and
> > ofcourse, troubleshoot ....and believe me, it is what constitutes
> > "thinking"
> > as well, not just the the wonderful wild trips of imagination which we all
> > love to indulge in .........and no, I don't consider myself grumpy little
> > scientist, please, my grad-schooling was not long ago for such luxuries
> > ......and the again some of your and my british colleagues may beg to
> > differ
> > from your view of the wonderful side of the coin....
> > .... my comments were a homage to all the lovely literature that gets
> > published, a lot of which is so unique that not even the good Mother
> >  Nature
> > will ever reproduce it ....
> >
> >
> >
> >
> > tom
> > >
> > > --
> > > Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London
> > WC1E
> > > 6BT
> > > (t) +44 (20) 76797264   (f) +44 (20) 76797805   (e)
> > > thomas.anderson from ucl.ac.uk
> > >
> > > _______________________________________________
> > > Methods mailing list
> > > Methods from net.bio.net
> > > http://www.bio.net/biomail/listinfo/methods
> > >
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
> Message: 5
> Date: Wed, 23 Jan 2008 00:29:47 -0200
> From: Felipe da Veiga Leprevost <leprevost from tecpar.br>
> Subject: Re: Gateway System
> To: methods from magpie.bio.indiana.edu
> Message-ID: <1a0d2f654c6f43aa50bf7c0a7a3c28e2 from webmail.tecpar.br>
> Content-Type: text/plain; charset="iso-8859-1"
>
>
>    Dear all,
>    " I want to use the gateway system of invitrogen to = minimize the
>    workload of subcloning our cDNAs in various expression = vectors.
>    Before
>    using the system I have to clone our cDNAs in an = appropriate pEntr
>    vector. I used pEntr1a for this but I do not get any = colonies on
>    kanamycin plates using homemade DH5alpha or Invitrogen = Top10 cells.
>    I
>    did remove the ccdB sequence before putting in our cDNA. = I also
>    tested
>    cutting out the ccdB sequence, religating the empty = vector and
>    transforming bacteria with this. I get only a very limited = amount of
>    colonies from transformed Top10 cells and none on our own = competent
>    DH5alpha."
>
>    Hi Koen,
>    What protocol are you following to enter the plataform? = Are you
>    making the BP reaction to clone your DNA into the entry vector?
>    Leprevost
>
>
> ------------------------------
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
> End of Methods Digest, Vol 32, Issue 21
> ***************************************
>


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