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Figure 4 | BMC Research Notes

Figure 4

From: Crude subcellular fractionation of cultured mammalian cell lines

Figure 4

Demonstration of the advantages of crude fractionation over whole cell lysis and scale up of the approach. (A) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well plate and subsequently lysed directly in NP40 or RIPA lysis buffer or processed according to the protocol in Figure 2 (Sequential). An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue or by Western blotting and probing with antibodies to various marker proteins of specific intracellular organelles as previously. (B) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish and then transfected the next day in triplicate with 400 ng of a construct encoding V5/6×-HIS tagged ERGIC-53. Cells were extracted 48 hours later either directly in RIPA buffer, directly in NP40 buffer or according to our extraction protocol. Proteins were then partially purified from each extract using Nickel resin (SIGMA #P6611) and eluted proteins were analyzed by 10% SDS PAGE followed by staining with Coomassie blue or by Western blotting and probing with the anti V5 antibody. (C) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish and then transfected the next day in with 1600 ng of pcDNA6/V5-His (Invitrogen) or constructs encoding WT or MUT COMP. Cells were harvested 48 hours later and extracted according to our protocol. (D) HEK293 cells were plated at a density of 4 × 105 cells per well of a 12 well dish or 8.4 × 105 cells per well of a 35 mm dish and processed according to the protocol in Figure 2. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by Western blotting and probing with antibodies to GAPDH, BiP and Lamin A.

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