Swine anti rabbit hrp conjugate

SDS-PAGE was carried out as described elsewhere, in 8%, 10% or 15% SDS-polyacrylamide gels. The gels were either stained with Coomassie blue R250 or, for western analysis, transferred onto PVDF membranes. Blocking was performed in 5% or 10% skimmed milk solution in PBS or PBST (PBS + 0.1% TWEEN-20). Primary antibodies were: mouse monoclonal anti-TAC102 (1:1000, GenScript), rat polyclonal anti-TAC102 (1:1000, Eurogentec), rabbit anti-Protein A (1:5000, Sigma), rabbit anti-myc (1:1000, Sigma), mouse anti-myc (1:1000, Sigma), mouse anti-EF1α (1:10000, SantaCruz), rabbit anti-ALBA3 (1:1000, [53 ]), rabbit anti-ATOM (1:10000, [54 (link)]), mouse anti-GFP (1:1000, Sigma), rabbit anti-GFP (1:1000, Sigma), rabbit anti-COXIV (1:1000), rabbit anti-LipDH (1:10000, [55 (link)]). Secondary antibodies were: mouse anti-rabbit HRP-conjugate (1:10000, Dako), rabbit anti-mouse HRP-conjugate (1:10000, Dako), swine anti-rabbit HRP-conjugate (1:10000, Dako), goat anti-rat 680 LT (1:10000, LI-COR), goat anti-mouse 800 CW (1:10000, LI-COR), goat anti-rabbit 680 LT (1:10000, LI-COR).

Cell extracts were mixed with Laemmli buffer to a final concentration of 5 million cell-equivalents/15 μl. Either one million or five million cell-equivalents (depending on the experiment) were loaded per lane on an SDS-polyacrylamide gel. The gels were resolved by 80-120V and transferred (wet transfer, 100V for 1h) onto PVDF Immobilon- FL transfer membranes (0.45 μm, MILLIPORE), followed by blocking (5% milk in PBS + 0.1% Tween-20 (PBST)) for 1 h at room temperature. Primary antibodies were incubated for either 1 h at room temperature or overnight at 4°C. Secondary antibodies were applied for 1 h at room temperature. All used antibodies were diluted in blocking solution. The following antibodies were used: mouse anti-TAC102 (1:1000, [24 (link)]), rabbit anti-HA (1:1000, Sigma-Aldrich), rabbit anti-ATOM40 (1:10000, [61 (link)]), rabbit anti-mouse HRP-conjugated (1:10000, Dako) and swine anti-rabbit HRP-conjugate (1:10000, Dako), rabbit peroxidase anti-peroxidase soluble complex (PAP) (1:2000, for detection of the PTP tag). After antibody incubation, the membranes were washed three times with PBST. A final wash with PBS was performed prior to detection of the protein with the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific). The Amersham Imager 600 (GE Healthcare) was used to visualize the protein bands on the blot.