Biology Reference
In-Depth Information
11. Thorough and patient resuspendation is an important step of
successful OB isolation. Use a blue pipette tip for rough resus-
pendation, and a yellow pipette tip to break the small pieces of
OBs.
12. This time, the OB layer is less solid due to lower sucrose con-
centration, but still solid enough to be collected with spatula.
If necessary, the left pieces of the layer could be collected
with a 200
μ
L pipette, but avoid drawing larger volumes of
the solution.
13. If the OBs have good integrity, in further centrifugation we
can collect the OB layer as usual. If the OB integrity is broken,
after centrifugation, we fi nd wide oil layer between water and
hexane phase and it is necessary to repeat all the isolation. The
integrity may be broken, only if the temperature during the
isolation is excessively increased. For example, hot sunny day
in a non-air-conditioned laboratory is not ideal for OB isola-
tion ….
14. Lower temperature is convenient for hydrophobic proteins.
15. You may use a comb with wider wells and load all the sample
into one well, but we have achieved best results with classic
10-well comb and by dividing the sample into four lanes.
16. The gel pieces dry out during the excision and dry gel pieces
tend to jump when touched by the scalpel. Avoid “gel jumping”
by wetting with a small drop of water.
17. Some agitators cannot reach so low temperature. In this case,
incubate the Eppendorf tubes in a container with ice, on a
planar agitator.
18. When the ZipTip pipette tip is once wet, avoid drawing in air
bubbles.
19. We used BioBasic-18 column (1 × 150 mm, 300
Å
pore size,
m fi lm thickness, Thermo Electron). Flow rate was
0.1 mL/min.
20. We used Thermo Electron LCQ Deca ion-trap mass spectrom-
eter. Instrumental parameters were as follows: capillary tem-
perature 280 °C; capillary voltage 30 V; spray voltage 4.5 kV;
sheath gas fl ow, 80 a.u.; and auxiliary gas fl ow, 5 a.u. Normalized
collision energy of 35 a.u. was used for ion fragmentation.
21. We used Bioworks 3.1™ software.
22. Chymotrypsin, known for its low specifi city, and even trypsin,
may cleave apart from their cleavage sites, also behind other
amino acids (for example besides histidine). Although the frag-
mentation spectra were reliable enough, we considered also
the peptides semi-tryptic or semi-chymotryptic.
5
μ
