Biology Reference
In-Depth Information
10. To determine the protein concentration prior to 2D DIGE, an
EZQ protein quantitation assay is performed. Prepare the
ovalbumin standards by dissolving the lyophilized protein in
the buffer used for solubilizing the sample (i.e., lysis buffer) at
2 mg/mL, then preparing serial twofold dilutions of this stock
down to 0.125 mg/mL.
11. Place a sheet of assay paper over the microplate and insert the
stainless steel backing plate into the microplate. Ensure that
the paper is securely in place.
12. Apply 1 mL of each protein standard and experimental sample
to individual wells of the microplate assembly. Include 1 mL of
buffer alone as a background control. Load each standard and
sample in triplicate. Allow samples to dry completely on the
paper. Remove the assay paper from the cassette and immerse
into a plastic staining tray containing 30 mL of methanol.
Incubate at room temperature for 5 min with gentle agitation.
After washing, air-dry the paper completely.
13. Place the assay paper into the EZQ protein quantitation reagent
(Component A) and agitate gently on an orbital shaker for
30 min. Cover the tray with aluminum foil to protect from
light. After staining, rinse the assay paper for 2 min in EZQ
rinse buffer. Repeat twice.
14. Image the assay paper while still wet using a Typhoon Trio
with 488-nm laser (excitation) and a 610-nm band pass emission
fi lter and PMT of 300 V. Quantify the fl uorescence, measured
as volumes of each spot corresponding to an experimental
sample, standard and background using ImageQuant TL (GE
Healthcare). Subtract the average volume of the triplicate
background spots from each of the standards and experimental
sample spots. Determine the average of the triplicate corrected
volumes for all standards and samples. Plot a standard curve of
the standards against the corresponding protein concentration
(0.125-2 mg/mL). Determine a line of best fi t based on a
quadratic equation, then calculate the sample concentrations
by solving the quadratic equation for
x
(i.e., the unknown con-
centration) whereby
y
= average volume of the triplicate spots.
15. Label 100 mg of membrane and cytosolic proteins separately
with 200 pmol of Cy5. Mix well by pipetting and pulse spin.
Incubate tube on ice for 30 min in the dark. Add 1 mL of lysine
to quench the labeling reaction. Mix by pipetting and pulse
spin. Leave the tube on ice for 10 min in the dark.
1. Prior to isoelectric focusing, rehydrate IPG strips by pipetting
evenly 450 mL of rehydration solution into the groove of the
reswelling tray without introducing bubbles. Peel off the plastic
backing that protects the IPG gel. Without touching the gel,
3.4. Two-Dimensional
Electrophoresis
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