Biology Reference
In-Depth Information
8. The pellet is now dried and solubilized in an appropriated sample
buffer for further analysis (
see
Note 18
). Alternatively, store
the pellet at −20 °C, for longer term preservation. Samples are
ready to use in protein quantitation and electrophoretic assays.
1. Calibration curve: Prepare several dilutions of standard protein
(BSA) containing volumes of 10, 20, 40, 60, 80, 100, and
120
3.3 Protein
Quantifi cation
L of BSA (0.2 mg/mL) into test tubes and make each up
to 800
μ
μ
L with distilled water. Use 800
μ
L of distilled water as
control.
2. To determine the sample concentration, assay a range of dilu-
tions (1, 1:10, 1:100, 1:1,000). Prepare duplicates of each
sample.
3. Add 200
L of dye protein reagent (Subheading
2.6
) to each
tube and mix well by gentle vortex-mixing (avoid excess
foaming).
4. Incubate the samples with the protein reagent at room tem-
perature for 30 min in darkness.
5. Measure absorbance at 595 nm of each sample and standards.
μ
3.4 One-Dimensional
Electrophoresis
1. Add 10
μ
L SDS-PAGE loading buffer to the protein sample
(2-10
μ
g of extracellular protein) in a fi nal volume of 30
μ
L
and heat samples at 95 °C for 5 min.
2. Prepare the resolving gel buffer, with 12 % (w/v) acrylamide
gel fi nal concentration, by mixing 2.5 mL resolving buffer,
4 mL 30 % acrylamide/Bis solution, 0.1 mL SDS solution, and
3.4 mL distilled water. Degas with vacuum for 15 min. Add
50
L TEMED, and pour the gel within a
7.25 × 10 cm, 1 mm thick gel cassette. Pour approximately
5 mL of this solution into the gel cassette and completely cover
the solution surface with isobutanol or water to obtain a fl at
front on top of the resolving gel. Leave at room temperature
until the solution is completely polymerized. Gently dry the
gel surface with blotting paper.
3. Prepare the solution for the stacking gel by mixing 1.25 mL of
stacking gel buffer with 0.67 mL acrylamide/Bis solution,
0.05 mL SDS solution, and 3.05 mL distilled water. Degas
with vacuum for 15 min. Add 25
μ
L APS and 5
μ
L TEMED
to the solution of the stacking gel and gently stir to obtain a
uniform solution. Pour about 2.5 mL above the resolving gel and
insert the well-forming comb into this solution (
see
Note 19
).
Polymerize the gel for at least 45 min at room temperature to
allow complete polymerization.
4. When polymerization is completed, remove the comb from
the stacking gel. Remove any spacer from the bottom of the
gel cassette, and assemble the cassette in the electrophoresis
μ
L APS and 10
μ
