Need help with IEF
hamel at ccu.umanitoba.ca
Wed Apr 21 09:26:43 EST 1993
Good luck trying to get pH 3- 10 in a MINI IEF, especially a tube gel!
We too studied a BASIC pI protein (Probasin, pI about 10- 10.5) ... we
found, through good advice from a visiting Norwegian, that Pharmacia's
Immobiline II Custom gradients carrier ampholytes, it's available for pH
between 3.5 and 10 (see cat.# 80-1255-70 to 80-1255-75). Also:
Immobiline Dry Strips (Cat.# 18-1018-61), pH 3- 10.5 are the best thing to
use, it's VERY difficult to get, let alone maintain, such a broad pH range
in a mini IEF tube ... still VERY difficult to get basic pH in mini tubes.
We even tried getting consistent results with ampholines between pH 8 and
11 (Pharmacia cat.# 80-1125-95 and 80-1125-96) .... Immobiline is GREAT ..
if you don't have access to a Multiphor II electrophoresis unit, try a
standard horizontal electrophoresis unit such as is used for agarose gels
.. simply place only enough buffer in the cathode and anode tanks to
cover the electrodes (use buffers recommended by Pharmacia for
Immobiline), then form wicks with Whatman 3MM, etc ... this also works ...
just be sure that gel platform DOESN'T get wet. The trick is to CAREFULLY
place the strip in between the mini PAGE plates at the top ... we used a
teflon sheet (or "slippery" whitish/clear plastic sheet that comes wrapped
around most PVDF or nylon membranes), wetted with sample buffer.
For GREAT SDS PAGE, see Shagger and von Jagow, Analaytical Biochemistry,
vol 166, pgs 368-379 (1987) ... Tricine-SDS PAGE for seperation of
Proteins in the Range from 1 to 100 kDa. This gel methos gives phenominal
resolution for 1- 100 kDa !!! Beats the heck out of ANY Laemmli type
system available. .... also Doucet, Murphy and Tuana, Anal.Biochem., vol
190, pgs 209-211 (1990) and Anal.Biochem. vol 168, pgs 265-271 (1988).
In article <1993Apr20.144753.2348 at gnv.ifas.ufl.edu> gab at gnv.ifas.ufl.edu writes:
>I have just begun to do some isoelectric focusing and have run into
>a snag. I am using BioRad's mini-protean cell, which uses capillary
>tubes for the IEF gels. The directions that came with the kit called
>for using 1.6% of ampholytes in the 5-7 pH range, and 0.4% of a broad-
>range ampholyte, pH 3-10. Since the protein I am working with has a
>pI of about 10, I decided to only use the broad range ampholytes, and
>bring their concentration up to 2%.
>The problem I have encountered is that when I checked the pH of the
>gel (by cutting it into 10 pieces, putting each in a small volume
>of .05 M KCL under vacuum for 2 hours, then checking the pH), I
>still only get a range of 4.3 - 8.
>I would appreciate any suggestions on how to get a gel with a pH
>gradient of 3-10. Thank you in advance- Beverly
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