Proteins on SDS-PAGE running down & then up!?!

Mavi Gozler mavigozler at yahoo_STOP_UCE_.com
Tue Apr 23 11:18:53 EST 2002


If everything had been going your way in running Laemmli (SDS-PAGE)
gels for a year, perhaps one or more your reagents need replacing.  Or
you recently replaced a reagent, and you made it up incorrectly (this
should be suspected whenever a problem like this arises).  Note that
your protein of interest (the 140 kDa band) should run about halfway
into the gel, which means you need to run about an 8% gel, if you are
using a non-gradient gel.

The failure to see proteins above 130 kDa transferred to your blot can
indicate one of at least two things:
(1) there is a degradation of high molecular weight proteins as a
group by proteases under the conditions in which you handle your prep.
This seems unlikely in your case, but should be considered.
(2) more likely is that your electrophoretic transfer conditions are
inadequate, which is bizarre if you've been successfully performing
the transfer for a year.  But assuming you have just begun doing this,
it is quite common to fail to move the groups/classes of higher MW
proteins either because time (even though it's overnight) and/or
constant voltage/current settings were improper.  You should always
see the same voltage or current measurements when doing the transfer
at start-up:  suspect you have made your (Tris-glycine) transfer
buffer incorrectly and stop transfer and re-make.  It is actually
possible to be quite theoretical about your voltage and time settings,
making somewhat decent assumptions about molecular mobility
(charge/mass), distance to be traveled, ionic strength of buffer, and
perhaps three or four other factors, all coming from you knowledge of
basic chemistry and physics and biology.  My old thesis advisor is the
SWAG-type experimentalist and thinks theoretical estimators waste
their time since they work with factors he believes are usually
assumptions, but I find I am on target more than off target.

If your Ponceau S stain is ever puzzling, always stain the gel with
your typical stain (Coomassie BB R-250 is better than any), which
helps you identify whether all proteins were truly transferred.

Also make sure that your denaturation (boiling) steps are adequate,
and that you have centrifuged boiled samples prior to gel loading!  So
many gels have been ruined by precipitated material (proteins, etc.).
Make sure that your total protein concentration is not too high before
adding your sample buffer concentrate, which avoids overloading and
precipitation.

On 19 Apr 2002 17:12:27 +0100, dear_natasha at yahoo.com (Natasha
Schokman) wrote:

>Hi,
>
>I have been running whole cell lysates from mouse 3T3
>fibroblasts using SDS-PAGE for over a year now.  But
>recently I have encountered the problem where for some
>unknown reason my pre-stained markers indicate that my
>148 kDa marker band (which is stained orange) is
>starting to migrate upwards above my 250 kDa marker
>band (which is stained blue).  The puzzling fact is
>that the rest of the markers below this point indicate
>that the rest of the gel has run fine as all the other
>smaller markers separate out fine.  


>Overnight transferring to a nitrocellulose membrane
>and Ponceau red staining indicates that there is no
>protein from ~130 kDa - top of the gel region. 
>However, the rest of the proteins from 130 kDa and
>smaller have separated properly.  Unfortunately for
>me, my protein of interest is 140 kDa!
>
>Any suggestion as to why this is happening would be
>appreciated!!
>
>Thanks,
>N@
>
>=====
>Friends are quiet angels who lift us to our feet when our wings have trouble remembering how to fly.
>
>__________________________________________________
>Do You Yahoo!?
>Yahoo! Tax Center - online filing with TurboTax
>http://taxes.yahoo.com/
>
>---




More information about the Cellbiol mailing list