Biology Reference
In-Depth Information
10. For the transfer of the IEF gel onto the SDS-PAGE gel, hold
the groove with the gel in contact with the edge of the glass
plate and slide the gel between the glass plates using a wire
suitably formed.
11. Overlay the IEF gels with agarose solution, add the running
buffer to the upper and lower (15°C) chambers, and start the
electrophoresis. For the entrance of the proteins into the SDS-
PAGE gel, apply low current (75 mA) for 15 min. When the
proteins have entered increase the current to 200 mA for
approximately 5-7 h.
3.6. Analysis
of Microdissected
Cells from Squamous
Cell Carcinoma of the
Lung and Bronchial
Epithelium Cells
1.
This protocol uses the information obtained from the optimi-
zation procedures described above.
2.
Harvest 5,000 cells of each (bronchial epithelial, small cell
carcinoma grade 2 cells and small cell carcinoma grade 3 cells)
by manual microdissection and incubate the cells in 50
μ
L of
lysis buffer each.
3.
For protein labeling, according to the previous optimization
(see Fig.
3
), reduce the proteins with 2 nmol TCEP and label
the proteins of the microdissected cells with 4 nmol Cy5 dye.
4.
For the preparation of the internal standard, reduce 5
g pro-
tein (reference proteome: lysate of a pool of grade 2 and grade
3 tumor tissue sections; see Fig.
4
) with 2 nmol TCEP and
label the proteins with 4 nmol Cy3 dye subsequently.
μ
5.
To each reaction add 5
μ
L of 1.4 M DTT to stop the labeling
reaction and add 5
μ
L of the ampholytes solution Servalyt,
pH 2-4.
6.
Mix the samples and analyze the mixture by 2DE as described
above.
1.
After 2DE, leave the gels between the glass plates and acquire
the images using the Typhoon 9400 scanner (Focal plane:
+3 mm; see Note 6). Choose the excitation wavelengths and
emission fi lters specifi c for each of the CyDyes according to the
Typhoon user guide. Low resolution pre-scans should be per-
formed to adjust the PMT for each channel to gain maximum
pixel intensities in the region of interest in the range 50,000-
80,000. Finally, scan the gel with a pixel size of 100
3.7. Image Analysis
μ
m.
2.
Before image analysis with the DeCyder software, crop the
images with ImageQuant TL (see Fig.
5
).
3.
For intra-gel spot detection and quantifi cation use the DIA
mode of the DeCyder software. Set the estimated number of
spots to 10,000. Apply an exclusion fi lter to remove spots with
a volume smaller than 30,000.
4.
Defi ne a representative gel from your experiment as master gel
and match spots between the different gels using the Biological
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