Biology Reference
In-Depth Information
12. Mix butan-2-ol:water (70:30) thoroughly for several min.
Then leave it to settle until two separated phases are formed
and take the upper phase (water-saturated butan-2-ol).
13. This SDS concentration is recommended for the upper cham-
ber of the Ettan Dalt Six for a correct migration of the proteins
to avoid spots that look like tears.
14. Weigh acrylamide inside the fume hood, wearing a mask.
Unpolymerized acrylamide is a neurotoxin, and care should be
taken to avoid skin contact.
15. Weigh SDS inside the fume hood, wearing a mask.
16. Avoid higher temperatures in the presence of urea to prevent
the carbamylation of proteins.
17. The pH of the lysate can be increased to pH 8.5 by the careful
addition of dilute sodium hydroxide (1 M). Test the sample
pH by spotting a small volume (0.2 mL) on a pH indicator
strip. If the pH increases more, add 1 N HCl to decrease it
again.
18. Protein assay RC DC Bio-Rad: pipette 25 mL of standards and
samples into clean and dry microfuge tubes. Add 125 mL of
RC reagent I into each tube and vortex. Incubate the tubes
for 1 min at room temperature. Add 125 mL of RC reagent II
into each tube and vortex. Centrifuge the tubes at 15,000 ×
g
for 3-5 min. Discard the supernatant by inverting the tubes
on a clean and absorbent tissue paper. Allow the liquid to
drain completely from the tubes. Add 127 mL of reagent A
(5 mL of DC reagent S to each 250 mL of DC reagent A) to
each microfuge tube and vortex. Incubate tubes at room tem-
perature for 5 min, or until the precipitate is completely dis-
solved. Vortex before proceeding to the next step. Add 1 mL
of DC reagent B to each tube and vortex immediately.
Incubate at room temperature for 15 min. Then, the values
for absorbance can be read at 750 nm. These will be stable for
at least 1 h.
19. Low-fl uorescence glass plates must be used for gel electropho-
resis using DIGE. They ensure the lowest background pixel
values of scanned images. Low-fl uorescence glass plates must
not be scratched as the scratches will appear on the image.
After casting, the gel(s) should be overlaid with water-saturated
butan-2-ol and left for 2 h at room temperature. The overlay
can then be removed and replaced with running buffer and left
until required. It is recommended that the gels are prepared at
least 1 day in advance to ensure reproducible results. Gels can
be stored at 4°C for up 2 weeks before use (as long as they do
not dry out).
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