Biology Reference
In-Depth Information
3. Place the ceramic strip holder on the IPGphor system aligned
in the direction indicated by anode and cathode regions. Run
the IEF parameters for 24 h run as follows: 30 V for 12 h
(step), 500 V for 1 h (step), 1,000 V for 2 h (gradient),
8,000 V for 2 h (gradient), and 8,000 V for 6 h (step).
This step involves reducing and alkylating the strip-bound pro-
teins, and transferring the IPG strip onto a preprepared 12 % (w/v)
acrylamide gel without a stacking gel encased in low-fl uorescent
glass plates ( see Note 27 ).
Transferring and Setting
IEF Strips onto SDS-PAGE
1. Incubate the IPG strip in equilibration buffer containing
65 mM DTT for 15 min in the dark with gentle rocking ( see
Note 28 ).
2. Incubate the IPG strip in equilibration buffer containing
135 mM IAA for 15 min in the dark with gentle rocking.
3. Rinse the strip for 5-10 s in 1.5 M Tris-HCl pH 8.8 contain-
ing 1 % (w/v) SDS.
4. Place the strip on the top of a 12 % (w/v) acrylamide gel, and
then add about 5 mL of warm IEF fi xing solution to secure
the strip ( see Note 29 ).
5. Once the IPG fi xing solution has solidifi ed, assemble the gel
apparatus and run the gels ( see Notes 30 and 31 ).
Once SDS-PAGE step is completed, the different proteomes under
comparison are visualized by scanning the gel at three wavelengths
(633, 532, and 488 nm) with a fl uorescent scanner according to
the manufacturer's instructions. The generated fi les are analyzed
by commercial software packages such as DeCyder 2D Software
(GE Healthcare Life Sciences) and DeCodon Software
(DECODON). Generally the literature [ 12 , 28 , 45 , 50 ] considers
a protein spot to differ signifi cantly between the two samples if the
abundance differs by greater than 1.5-fold with a P -value of less
than 0.05 following Student's t -test.
Gel Scanning and Software
Analysis
The DIGE analysis reveals which protein spots display differing
abundance, but peptide mass spectrometry is required to identify
these proteins. As CyDye-labelled proteins cannot be seen with the
naked eye and DIGE gels are relatively of low abundance it is
advantageous to use preparative gels for mass spectrometry identi-
fi cation ( see Note 32 ).
1. Match the spots of interest carefully from the DIGE gel with
its corresponding spot on the preparative gel, excise the spots,
and place them into the wells of the 96-well plate.
2. To each well add 50
Extraction and Digestion
of Proteins That Differ
in Abundance on DIGE
L of destain solution to each excised gel
plug, cover the 96-well plates, and shake on orbital rocker at
700 rpm for 30-45 min.
μ
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