Nuclear extract preparation from cells was carried out as previously described [17].
For the screen presented in this report, one hundred strains were chosen at random for extract preparation from the Hartwell collection of temperature sensitive (ts) strains (27).
The medium was also supplemented with 0.125 mL Vitahop-LactoStab® (hop extract preparation, from BetaTec Hop products, Germany) before inoculation to prevent microbial infections.
While the high sensitivity of biochemical assay and the wide range of activities displayed by GBMs and AGs (~ 300-fold) support the use of MGMT activity to further stratify risk of progression, the nature of the biochemical assay, requiring extract preparation from intact tissue and the use of a radioactive substrate that is not commercially available, make it unsuitable for routine clinical use.
Averaged 2-D polypeptide maps were obtained using gels of three independent preparations, each from a different batch of plants (gels obtained with different root tip extract preparations isolated from different batches of plants were very similar; data not shown).
For RNA isolation or cell extract preparation for western blot analysis, tissues from placenta, decidua, spleen, thymus and lymph nodes (inguinal, axillary, mesenteric and iliac) were carefully washed with cold sterile PBS, pH 7.40, snap-frozen, and kept at −80 °C until use.
The mechanism of action of herbal drugs and their extract preparations, which differ in many respects from that of synthetic drugs or mono substances, can be characterized as a polyvalent action and interpreted as additive or, in some cases, potentiating.
The protocol for cytoplasmic and nuclear extract preparation was adapted from previously published procedures [38], [39].
For extract preparation, 0.5 mg tissue from tepals, anthers and stigma (collected at three different stages) was crushed with a micropestle in 700 μl Tris HCl (50 mM, pH 7.5 containing 1 M NaCl), and incubated for 10 minutes on ice.
