Immunoprecipitation of Presenilin-1 from Insect Cells

Co-Immunoprecipitation was carried out as described previously^{104 (link),105 (link)}. Infected insect cell membranes were dissolved in 1% CHAPSO-HEPES buffer (25 mM HEPES, pH7.4; 150 mM NaCl; 1% CHAPSO w/v; Buffer A) and diluted to 1 mg/ml protein concentration, using the same buffer. Soluble membranes were incubated with PS1 antibody Ab14 (rabbit polyclonal raised against 1–25 amino acids of PS1) overnight at 4 °C under gentle agitation. Protein-A Mag Sepharose (GE, UK 28951378) was used to harvest protein antibody complexes and eluted in LDS sample buffer (Invitrogen, USA NP0007) at 37 °C for 15 minutes for SDS-PAGE and Western immunoblotting.

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Khan I., Krishnaswamy S., Sabale M., Groth D., Wijaya L., Morici M., Berger I., Schaffitzel C., Fraser P.E., Martins R.N, & Verdile G. (2018). Efficient production of a mature and functional gamma secretase protease. Scientific Reports, 8, 12834.

Publication 2018

Protein-A Mag Sepharose LDS sample buffer

Amino acids Antibody Buffer Cell membranes Chapso Co immunoprecipitation Hepes Insect Membranes Ml 1 protein Nacl Protein Protein a sepharose Rabbit Sds page

Corresponding Organization : Curtin University

Other organizations : Edith Cowan University, University of Western Australia, European Molecular Biology Laboratory, Discovery Centre, University of Toronto

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Variable analysis

independent variables

CHAPSO concentration (1% w/v)

dependent variables

Protein-antibody complex formation
Protein elution in LDS sample buffer

control variables

HEPES buffer composition (25 mM HEPES, pH 7.4; 150 mM NaCl)
Protein concentration (1 mg/ml)
Incubation conditions (overnight at 4 °C under gentle agitation)

controls

Positive control: Protein A Mag Sepharose used to harvest protein-antibody complexes
Negative control: Not explicitly mentioned

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