Contributed by James V. Anderson and Charles L. Guy, The University of Florida, Institute of Food and Agricultural Sciences
BiPs (Immunoglobulin Heavy Chain Binding Proteins, also known as GRP78; Glucose Regulated Proteins) are ER-luminal proteins and members of the HSP70 family.1 BiPs are associated with the transport and folding process of proteins involved in the secretory pathway. BiPs and many other HSP70 proteins can easily be partially purified by affinity chromatography with ATP-agarose. However, due to BiPs ability to bind proteins, we have found it associated with other contaminating proteins over a broad range of salt gradients when using ion-exchange chromatography, and over a broad molecular weight range when using size-exclusion chromatography. Here we report a rapid method for the further purification of affinity selected BiP suitable for amino acid sequencing and antibody production.
Native extracts from 3 week old spinach leaf tissue were taken through 40 and 80% ammonium sulfate (AS) precipitation. The resuspended 80% ammonium sulfate pellet was refractionated by an additional 1 M and 3 M ammonium sulfate precipitation. The resulting 3 M pellet was resuspended in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.1 mM DTT (Buffer A) and then dialyzed overnight against two changes of buffer A plus 3 mM MgCl2 (Buffer B). The dialyzed sample was centrifuged at 20,000 x g to remove insoluble material. The clarified sample was loaded onto an ATP-agarose affinity column (1.5 x 10 cm bed volume) equilibrated with buffer B at a flow rate of 0.5 ml/min. S