Biology Reference
In-Depth Information
8. Wash the pellet in 1.2 mL of 90 % acetone (diluted with dis-
tilled water). Pipette up and down to disaggregate the pellet.
Centrifuge 5 min at 5,000 ×
g
and 4 °C to pellet down the
proteins. Discard supernatant.
9. Air-dry the pellets until the acetone is completely eliminated.
Pellets may retain some water. This will help the pellet solubili-
sation (
step 9
).
10. Resuspend pellet in RS buffer. A complete pellet solubilisation
may take up to 3 h in a thermal shaker (35 °C and 750 rpm),
being also some occasional pipetting needed (
see
Note 3
).
11. Centrifuge a maximum speed and room temperature for 5 min
to remove insoluble particles. Transfer the supernatant to a
new tube (
see
Note 4
).
1. Prepare Standard Working Reagent (SWR): Mix 100 vol. of
regent A with 2 vol. of reagent B. The solution is stable at
room temperature for 1 week.
2. Add BSA standards to an ELISA plate. 0, 1, 2, 3, 4, 6, 8, 10,
15 and 20
3.2 Protein
Quantitation
g of BSA are using for the calibration curve (three
technical triplicates are advised).
3. Add 2
μ
L sample to each well.
4. Add 200
μ
L of SWR to each well. Shake the plate gently.
5. Close the plate with a cap or aluminum foil to minimize evapo-
ration. Incubate the plate 20 min at 60 °C (in an oven) or
40 min at 45 °C in the plate reader. Read absorbance at
562 nm. Use the spectrophotometer bundled software or
spreadsheet for calculating concentrations (
see
Note 5
).
μ
1. Mix 40-80
μ
g of protein (minimum recommended 50
μ
g), 5
μ
L
3.3 Protein
Fractionation by
SDS-PAGE
of 5× Laemli buffer, and water to a fi nal volume of 25
L (this
may vary in function of the well size/electrophoresis system).
μ
2. Perform a standard SDS page fractionation in a 12.5 % acryl-
amide gel (maximum thickness 1 mm). Before loading the
protein, mark a line 1 cm below the staking-resolving inter-
phase. Load the samples leaving a blank well between them to
avoid contaminations (
see
Note 6
) (Fig.
1
).
3. Run the gel at 80 V, constantly, until the bromophenol blue
reach the marked line.
4. Stop the electrophoresis and immediately transfer the gel to a
plate with Coomassie staining solution. Incubate in an orbital
shaker for 30 min (recovery of the tryptic peptides is dramati-
cally reduced in overfi xed gels).
5. Destain the gels in destaining solution for 80 min, replace
destaining solution every 20 min.
