Biology Reference
In-Depth Information
5.
Add water dropwise until almost 1 mL. Mix by inverting
several times to dissolve the urea. Make up to fi nal volume
(1 mL) with water.
6.
We use Strip Holder Cleaning Solution from GE Healthcare.
7.
We use PlusOne DryStrip Cover Fluid from GE Healthcare.
8.
The urea may dissolve slowly. If needed, heat the solution
moderately (do not boil) and allow to cool before adding DTT
or iodoacetamide.
9.
Leave the weighed DDT and iodoacetamide in beakers at 4°C
while EB is being mixed or cooled.
10. Due to the heterogeneous nature of biological samples, there
are many varied protocols for preparing sample lysates. The
procedure described here is used by our laboratory for most
tissues samples, cell pellets, and subcellular organelles. We have
also included a few references for preparing unique samples
such as adipose tissue (
7, 8
), membrane proteins (
9
), and post-
synaptic densities (
10, 11
).
11. DNase may be added if samples are still very viscous after
sonication.
12.
We use the 2D Clean-Up Kit from GE Healthcare. Alternatively,
a chloroform-methanol precipitation method may be used (
12
).
13. With our microcentrifuges, a “quick-spin” consists of 5-10 s
where the speed does not reach more than 1,699 ´
g
.
14. If the pellet does not fully dissolve in 40
μ
L, add another 10
μ
L
of 2× ubiquitin conjugation buffer and 10
μ
L of water until it
does.
15. If necessary, samples may be incubated with NEM for 3 h or
more on ice.
16. We use strip holder cleaning solution and a cleaning brush
from GE Healthcare. Scrub the holder using the brush and
rinse thoroughly with pure water.
17.
Prehydrate 24-cm strips for 2 h in 200
L of rehydration
buffer (see Subheading
2.4
, item 2). Account for this volume
when preparing samples for isoelectric focusing (i.e., 450-
200
μ
L rehydration buffer).
18. To remove bubbles once the strip has been applied, gently tap
the back of the strip with a clean 200-
μ
L = 250
μ
μ
L tip. If bubbles remain,
pull the strip up and try again.
19. If strips run overnight or fi nish before you are prepared to start
the second dimension, a step 5 of 100 V for 24 h may be added
at the end of the protocol as a “hold” step to keep the strip
focused. This can be stopped once you are ready to proceed to
the next stage.
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