Five picomoles of RNAP and 25 pmol of HelD, T4 DNA ligase (TaKaRa) or MLV reverse transcriptase (Promega), respectively, were used. Proteins tested for mutual interactions were incubated for 15 min at 30°C in 10 μl in the storage buffer. After incubation samples were mixed with 3 μl of Native PAGE 4× Sample buffer (Invitrogen) and loaded onto the Native PAGE 4-16% Bis-Tris Gel (Invitrogen) and electrophoresed. The gels were subsequently Coomassie stained.
Native page 4 sample buffer
Manufactured by Thermo Fisher Scientific
Native PAGE 4× Sample buffer is a concentrated solution designed for use in native polyacrylamide gel electrophoresis (Native PAGE) applications. It is used to prepare protein samples for analysis while maintaining their native, non-denatured state.
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2 protocols using native page 4 sample buffer
1
Native PAGE Protein Interaction Assay
2
Lipidation Analysis of APOE Protein
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Lipidation of APOE in conditioned medium was assessed using native gel analysis. 20 µl of conditioned medium and 2 μl of HA-tagged APOE protein purified from bacteria was added to 6.25 l NativePAGE 4× sample buffer (Invitrogen BN2008) and run on a NativePAGE mini gel (Invitrogen BN2112BX10) in light and dark cathode buffer and anode buffer made with NativePAGE running buffer (Invitrogen BN20001). Gel was transferred to an Immobilon-P PVDF membrane (Millipore IPVH00010). Membrane was blocked with Pierce StartingBlock Buffer (Fisher Scientific EN37543) for 30 min and incubated overnight with primary antibody. The following day, membrane was washed three times with TBS with 0.1% Tween-20, and incubated for 1 h in StartingBlock Buffer with HRP-conjugated secondary antibody. SuperSignal West Dura Chemiluminescent reagents (Fisher Scientific PI34076) were used to detect HRP, and membrane was imaged on an Azure c600 system.
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