D.K.,
I did not mean to hurt your feelings, although I was a bit irritated by the
abundance of vague or sometime condescending phrasing in your message ("If
you don't have[a lot of protein] ..., save yourself time and send it to
mass-spec-based sequencing" - which we don't have in our university; "They
all work and if _some_ does not work, the main problem is elsewhere"; "If
you have enough common sense..."; "...if you are that paranoid...", etc.)
Here are excerpts of another message I got in reply to my post. I believe
these recommendations might be useful to somebody:
"I have been routinely sequencing bands from gels for about the past 9
years.
...
We use commercial pre-cast ... tris-tricine gels.... The lower pH
of tris-tricine gels has been held to cause less potential modification than
tris-glycine gels. ...cast [gels] the day before use and leave to polymerize
overnight. ... Many suggest adding ..thioglycolic acid.. to the cathode
buffer to act as a free-radical
scavenger (about 0.1mM). I never pre-run the gel.
...
Don't heat your sample at 95celcius prior to loading on the gel. It is
better to heat at about 56celcius for 10minutes or 37celcius for 30mins.
This is to reduce the chance of n-terminal modification.
...
Always use sequencer grade PVDF, we use ABI ProBlott. These are thicker
membranes and are more retentive of protein. As an example protein will go
through several sheets of Immobilon-P during electrotransfer, whereas only
very high loads get through ProBlott. Retentiveness is important as use of
low grade PVDF will give poor repetitive yields on sequencing (there is
literature support for this)...
I always use 10mM CAPS at pH11 (dilute from 100mM stock) and 10% methanol
... which gives good results. Don't use glycine-based buffers. ... Transfer
...
[at] 1.5 - 3.0mA/cm2 for 1-2hr for tank blotting and 0.8-2.5mA/cm2 for 30
mins
with semi-dry blotting. ..."
I think these recommendations are not just a cookbook recipe. The message
contains details behind certain important points that help me understand
better what I am going to do. I feel quite comfortable following these
recommendations, because if this method does not work in my hands I will
assume that it is rather because of the nature of my protein than because of
a wrong membrane or the wrong gel. I hope you understand what I mean.
- Emir
"D.K." <dk at no.email.thankstospam.net> wrote in message
news:9sa1g7$aru$1 at news.doit.wisc.edu...
> In article <AWSF7.78$N4.3944 at news.uchicago.edu>, "Emir"
<REMOVEem at unforgettable.com> wrote:
> >Look, DK, I appreciate your effort to reply, but I thought it was clear
that
> >I am not looking for the common sense recommendations (pre-run the gel,
> >rinse the blot,... wear gloves) a more or less experienced biochemist
would
> >be aware of. I welcome feedback from people with first-hand experience
and
> >successful solutions.
>> I thought that's exactly what I provided (based on first hand experience).
> Lets summarize: Have plenty of protein, use any PVDF brand, pre-run for
> 5-10 min only, transfer in anything you want (tris/glycine, CAPS,
carbonate,
> borate), but if you use tris/glycine wash membrane very, very thoroughly.
>> >Sequencing costs money ($200 for 5 cycles in our case) and it is not
> >desirable for us to pay for attempts doomed to fail . Just-do-it
philosophy
> >(I understand this in your understanding is an alternative to a "cookbook
> >philosophy" :-)) is not working all the time.
>> No, my understanding of an alternative to a cookbook philosophy is
> understanding exactly what you are doing and why and adjusting things
> you are doing based on this knowledge rather than on religious beliefs.
>> An example: that glycine thing. The same people who told you to not
> run transfer in Towbin buffer do their gels by Laemmli. In other words,
> they do transfer from gels containing glycine.
>> >The staff at the sequencing facility specifically pointed out that high
> >glycine backgrounds due to using glycine transfer buffers and blocking of
> >sequencing due to protein modification during PAGE are very common causes
of
> >failure. As for the quantity of the protein, I was told 50 pmol of my
~50kDa
> >protein (i.e. ~50ng/band) would be enough.
>> Yes, 50 pmol is OK for Edman degradation, but - sorry - for a 50 kDa
protein
> 50 pmol is 2.5 micrograms, which is exactly what I meant - A LOT. With 50
ng
> you can still do it just fine by mass-spec.
>> DK
