Contents for 2D Gel Electrophoresis
Immobilized pH Gradients
Immobilized pH gradient isoelectric focusing
technology is a major step forward in the field of isoelectric
focusing. Using small, pH gradient forming acrylamido-acids and
bases, pH gradients are formed that have the benefit of being stable
(do not diffuse or experience cathodal shift). Additionally, the
gradients can be tailor-made to fit individual needs of length and
pH range. This gives the scientist extraordinary power to zoom in
over extremely narrow pH ranges to separate and identify proteins
with extremely high resolution.
The PCL utilizes Immobilized pH gradient technology
(IPG DryStrip) provided by Amersham Biosciences. The lab has two
IPGPhor focusing units and routinely handles strips of either 7 cm
(mini-gel) or 13 cm (large format) in length and a variety of
commercially available pH ranges. As in all Isoelectric focusing
protocols, sample preparation is of utmost importance to the overall
success of the experiment. Additionally, as all samples are unique,
individual attention may be required to facilitate the successful
outcome of this type of work therefore we require that scientists
contact the PCL in advance of submitting samples for this analytical
service.
Sample Preparation and Submission
Samples for IEF can be submitted as liquid samples
or dry (lyophilized) powders. Liquid samples must be relatively free
of non-protein contminants. Salts, in particular, must be eliminated
or at very low concentrations. Ionic detergents must be avoided as
well as any ionic buffering agents. These materials must be removed
from samples either by dialysis or precipitation. Keep in mind,
however, that these methods can result in sample loss.
Lyophilization requires prior dialysis so that salts are not
concentrated.
The PCL has run validation experiments to illustrate
the effects of salts on the performance of the 2D gel (see FAQ).
In addition, we have run validation experiments on
precipitation techniques to help you visualize the degree of sample
loss when comparing acetone and TCA methods against non-precipitated
methods (see FAQ). Molecular weight cutoff membranes may also be
used but be mindful that proteins may bind.
FAQ's and Gel Examples
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What effects will salt have on my 2D gel?
In general, samples will streak and some proteins may be lost.
We recommend that liquid samples contain no more than 50 mM TOTAL
salt concentration (buffers & salts).
Gel A: 0 mM NaCl, Gel B: 10 mM
NaCl, Gel C: 50 mM NaCl.
-
What is the maximum amount of protein that I
can run on a 2D gel?
We recommend a maximum of 25-500 ugm for Coomassie staining
and 50-100 ugm for silver staining of a complex cell lysate.
Gel A: 60 ugm, Gel B: 180 ugm,
Gel C: 300 ugm.
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How can I concentrate my sample and reduce
sample loss?
All samples are unique and require special attention. No one
answer will suffice. We routinely use Acetone precipitation as we
think it yields better recoveries and maintains the protein
profile better than TCA or spin conentrator procedures. You must
remember that re-solubilization is a key point of sample loss. In
particular, hydrophobic proteins will be difficult to resolubilize
regardless of method used. You might consider lyophilization or
the SDS protocol we use for O’Farrell gels. (Call to discuss your
needs).
Gel A: No precipitation, Gel B: TCA precipitation,
Gel C: Acetone precipitation.
-
Which staining procedure should I use?
We recommend Coomassie staining for high abundance samples and
those that will be used for in-gel digestion and mass
spectrometry. It typically is considered useful for protein above
50 ng per spot. Silver staining is nearly 10-50 times more
sensitive and is useful for detecting very small amounts (~1-5 ng
per spot) of protein and displaying a larger number of proteins in
the gel. However, Coomassie staining is the best stain for easier
identification of proteins using in-gel digestion and mass
spectrometry. (See comparison below).
Gel A: Silver stain, Gel B: Coomassie stain. Both gels are loaded with equivalent amounts of
E. coli cell lysate.
-
Do samples dissolved in SDS really work on
O’Farrell style tube gel IEF?
Yes. We are surprised, too. Following a suggestion from Kati
Kiss (Dept of Microbiology, HSC, TAMU) we tried it on our
difficult-to-dissolve samples and found outstanding results.
Indeed, some folks think they see more spots on the SDS-treated
sample. See below.
Gel A: PC6 cell lysate on SDS
tube gel ala O’Farrell (pH 3.5-10NL custom gradient), Gel B:
Urea sample of same PC6 cell lysate on IPGDryStrip (pH 3-10NL).
Equivalent amounts of protein were separated and stained with
silver.
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