Biology Reference
In-Depth Information
coverage cutoff of 0, an annotation cutoff of 55, and a GO
weight of 5 although this can be adjusted to the analysis
requirements [
44
]. Run InterProScan and merge InterProScan
GOs to annotation. Then, run GO-enzyme code mapping. At
this stage, protein sequences are annotated and ready to apply
functional the analysis tool using B2G software (
see
Note 35
).
4
Notes
1. Select a container twice the size of the fi nal volume you want
to prepare. Measure out approximately 90 % of the fi nal
required volume of water, e.g., 900 mL for a fi nal volume of
1,000 mL, and while stirring the water, add the solid compo-
nents of the medium and stir until completely dissolved and
after adjusting pH, add additional water to bring the medium
to the fi nal volume.
2. Plant tissue culture media are generally sterilized by autoclav-
ing at 121 °C and 1.05 kg/cm
2
(15-20 psi). The time required
for sterilization depends upon the volume of medium in the
vessel since the time required for the liquid volume to reach
the sterilizing temperature (121 °C) is different.
3. Distribute the medium in Petri dishes (20 ml in each) when its
temperature is about 45 °C and when the medium solidifi es
sealing the plates with Parafi lm
®
to avoid dehydration.
4. Please check the list of compatible chemicals and potential
interfering chemicals typically found in the protein extraction
buffer.
5. Do not freeze vials with stock solution more than once.
6. It is best to prepare this fresh each time.
7. TEMED accelerates the decomposition of APS molecules
into sulfate-free radicals and these, in turn, initiate the
polymerization.
8. Simple method of preparing running buffer: Prepare 10×
native buffer (0.25 M Tris, 1.92 M glycine, and 1 % SDS).
Weigh 30.3 g Tris, 144 g glycine, and 10 g of SDS, mix, and
make it to 1 L with water. The pH of this solution should not
be adjusted and store at room temperature. Dilute 100 mL of
10× native buffer with 900 mL of distilled water.
9. The choice of the most appropriate rehydration solution for the
sample depends on its specifi c protein solubility requirements.
Urea solubilizes and denatures proteins, unfolding them to
expose internal ionizable amino acids. For solubilization of the
more hydrophobic proteins, use thiourea/urea [
45
].
10. LTQ Orbitrap (Thermo Fischer Scientifi c); XCT ion trap
(Agilent Technologies). Search engines: Proteome Discoverer
