The modifications are fixed before fertilization--some by the mother, some by the father--switching so-called imprinted genes either on or off during development.
Given a parent ion mass and a database, once the allowable PTMs are specified, the number of peptides along with possible types of modifications are fixed.
As variable modifications, oxidation of histidine, tryptophan or methionine, dioxidation of tryptophan and tryptophan to kynurenin modification were allowed, fixed modification was set for carbamidomethylation of cysteine.
For EGFP and RsbW levels proteins were extracted from 100 mL of stationary cultures grown for 12 days at 4°C using the sonication-based method with a slight modification; cells were fixed in 1 : 1 volume of ice cold 1 : 1 (v/v) methanol/ethanol mixture for 10 min at −20°C before centrifugation.
Modifications included were: fixed; carbamidomethyl cysteine and variable; oxidation of methionine.
ERGs were performed as described in Fabian-Fine et al. (2003) with the following modifications: flies were fixed using Elmer's non-toxic Glue-All.
ChIP was performed as described (Pidoux et al., 2004) with the following modifications: Cells were fixed in 1% PFA/15 min for H3K9me2 or Stc1-FLAG ChIP, or 3%PFA/300 min for Swi6 ChIP.
Apoptosis (TUNEL assay) and proliferation were assayed in-well of an eight-well chamberslide as in [ 20] with the following modifications: cells were fixed in 4% paraformaldehyde and Ki-67 was obtained from Vector Labs, Burlingame, CA, USA, and detected by Alexa Fluor 568 goat anti-rabbit IgG secondary antibody (Molecular Probes/Invitrogen).
In situ hybridization followed previous described protocol [4] with the only modification that embryos were fixed following incubation within 2 minutes.
