The notes describe the process, but the chapter itself describes three methods for extracting protein: a total protein extraction, a centrifugation-based subcellular fractionation protocol, and a detergent-based subcellular fractionation protocol. Most protocol steps have additional comments from the author...
The protein yield with this protocol was about 2.0-2.8 mg/ml depending upon the cell types. FIG. 4 (SDS-PAGE) shows a comparison of proteins extracted by repeated sonication and proteins extracted by solution A coupled with protein extraction cartridge of present invention. Lane 1, proteins ...
Protocol summary for the 1-Step Human Coupled IVT Kit. Simply add the appropriate template to a mixture of HeLa cell lysate, accessory proteins, and reaction mix and incubate at 30°C for 90 minutes for protein yields up to ...
from these cells, one would therefore expect a size distribution in which 90% of the objects are 4.6 Mbp, whereas the remaining 10% would contain DNA at an amount of between 4.6 and 9.2 Mbp, depending on how far genome replication in the cell had proceeded at the time of DNA extraction...
Digitonin extraction protocol was adapted from ref. 98. Cytosolic and membrane-associated RNA fractionation experiments were performed in 80% confluent 12 multiwell plates. Cells were washed twice in ice-cold PBS and incubated for 5 min in 200 µl of digitonin extraction buffer (0.03% of ...
which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein syn...
Preparing Cell Lysates To detect your fusion protein by Western blot, you will need to prepare a cell lysate from transfected cells. A sample protocol is provided below. Other protocols are suitable. To lyse cells: Wash cell monolay...
Metformin as a first-line clinical anti-diabetic agent prolongs the lifespan of model animals and promotes cell proliferation. However, the molecular mechanisms underlying the proliferative phenotype, especially in epigenetics, have rarely been reported.
also used this protocol for lysates and culture supernatants of other human cell lines such as HEK293T. The protocol below describes the specific steps for the culture supernatant of A431 cells, a human cell line derived from epidermoid carcinoma, selected as a typical adherent mammalian cell ...
Cell counting was carried out on an automated cell counter (Countess, Invitrogen). FASP-based Protein Processing Aliquots of lysates containing 100 μg of total protein were processed according to the multi-enzyme digestion FASP protocol (18). Briefly, protein lysates were depleted from the ...