Blue Native Page Polyacrylamide Gel Electrophoresis (BN-PAGE)

Cell extract was diluted 6:1 (v/v) with Coomassie Brilliant Blue G-250 (5%; w/v) at 4 °C.

Fifty mL of 20x NativePAGE™ running buffer (Invitrogen) and 50 mL 20x NativePAGE™ cathode additive (Invitrogen) were added to 900 mL deionized H₂O for preparation of 1x NativePAGE™ cathode buffer at pH 7.5 and 4 °C. Cathode buffer consists of 15 mM Bis-Tris, 50 mM trycine and 0.02% (w/v) Coomassie Brilliant Blue G-250.

Fifty mL of 20x NativePAGE™ running buffer (Invitrogen) was added to 950 mL of deionized H₂O for preparation of 1x NativePAGE™ anode buffer at pH 7.5 and stored at 4 °C. Anode buffer consists of 50 mM Bis-Tris.

Lipogenic multienzyme complex in crude cell extract was separated by BN-PAGE²⁸. Briefly, a precast native gradient gel (Native PAGE™ Novex Bis-Tris Gels, Invitrogen) with 3–12% gel gradient were used. This gel provides molecular weight resolution from 15 kDa to 10 MDa. The BN-PAGE was performed by Xcell SureLock™ Mini Cell (Invitrogen). A 300 mL of cathode buffer was added into the upper buffer chamber followed by the addition of 600 mL of anode buffer into the lower buffer chamber. Twenty µL of cell extract containing approximately 80 µg protein were loaded to each well. Twenty µL standard protein NativeMark™ unstained protein standard (Invitrogen) consisting of IgM hexamer (1236 kDa), IgM pentamer (1048 kDa), apoferritin band 1 (720 kDa), apoferritin band II (480 kDa), β-phycoerythrin (242 kDa), lactate dehydrogenase (146 kDa), bovine serum albumin (66 kDa) and soy trypsin inhibitor (20 kDa) was loaded into the well of the same gradient gel. All steps were performed at 4 °C. The molecular weight of the proteins that resolved in the gradient gel were estimated based on exponential plot of molecular weight of standard proteins against relative migration distance (R_f) of standard proteins.

Electrophoresis was performed at 4 °C, 100 V for 3 h and subsequently run overnight at 150 V.

After the electrophoresis, the gel was sliced into 10 groups with an approximate length of 8 mm each, and designated as group 1 (upper part of the gel) to group 10 (lowest part of the gel).

In-gel trypsin digestion²⁹ was performed using trypsin profile IGD kit for in-gel digests (Sigma-Aldrich). Each of the gel bands (1 to 10) was further sliced separately into smaller pieces and transferred into silicone tube. Two hundred µL of destaining solution (400 mM NH₄HCO₃ in 40% acetonitrile) was added into the tubes and incubated at 37 °C for 30 min. This step was repeated twice.

Reduction of disulphite bonds of the proteins was done by the addition of 200 µL 10 mM dithiothreitol (DTT) with incubation at room temperature for 30 min. The DTT solution was then removed. Two hundred µL 55 mM iodoacetamide was then added into the silicone tubes for alkylation of the proteins and incubated in the dark at room temperature. The solution was then removed and replaced with 400 µL of washing solution (50 mM NH₄HCO₃) and incubated at room temperature for 15 min. This step was repeated twice. Then, 400 µl acetonitrile (100%) was added into the silicone tubes to dehydrate the gel pieces and incubated for 10 min at room temperature. The supernatant was removed and the gel pieces were dried by incubation at 50 °C for 5 min.

Next, 20 µL (0.4 µg trypsin) trypsin (1 mM HCl and 40 mM NH₄HCO₃ in 9% of acetonitrile) was added into the silicone tube and incubated at room temperature for 5 min. Then 50 µL of 40 mM NH₄HCO₃ in 9% acetonitrile was added in the tubes and incubated overnight at 37 °C. The digested peptides were then transferred into new silicone tubes and dried again in vacuum centrifuge. The dried peptides were stored at −20 °C before tandem mass spectrometry analysis was performed.